A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence

Riham Ayoubi; Maryam Fotouhi; Charles Alende; Kathleen Southern; Carl Laflamme; NeuroSGC/YCharOS/EDDU collaborative group; ABIF consortium

doi:10.12688/f1000research.145146.2

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Revised

A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence

[version 2; peer review: 2 approved]

Riham Ayoubi¹, Maryam Fotouhi¹, Charles Alende¹, [...] Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

Riham Ayoubi¹, Maryam Fotouhi¹, [...] Charles Alende¹, Kathleen Southern¹, Carl Laflamme ¹, NeuroSGC/YCharOS/EDDU collaborative group, ABIF consortium

PUBLISHED 09 Jul 2024

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada

Riham Ayoubi
Roles: Investigation, Methodology, Validation

Maryam Fotouhi
Roles: Investigation, Visualization

Charles Alende
Roles: Investigation, Visualization

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Supervision, Validation

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the YCharOS (Antibody Characterization through Open Science) gateway.

Abstract

Protein phosphatase 2A is a serine/threonine phosphatase with activity dependent on an associated regulatory subunit, serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D). PPP2R5D is the δ isoform in the B56 family of regulatory subunits. Abundantly expressed in the brain and involved in a broad range of cellular processes, PPP2R5D plays an essential role in modulating key neuronal pathways and signalling. Pathogenic mutations in the PPP2R5D gene are linked to clinical symptoms characterized by neurodevelopmental delay, intellectual disability, and autism spectrum disorders. The etiology of these genetic disorders remains unknown, which can partly be due to the lack of independently characterized antibodies.

Here we have characterized six PPP2R5D commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords

Uniprot ID Q14738, PPP2R5D, Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform, antibody characterization, antibody validation, Western Blot, immunoprecipitation, immunofluorescence

Corresponding author: Carl Laflamme

Competing interests: For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific.

Grant information: The grant was from a Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). The study was also funded in part by the Simons Foundation Autism Research Initiative (SFARI). RA is supported by a Mitacs fellowship. 
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Ayoubi R et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Ayoubi R, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved]. F1000Research 2024, 13:1 (https://doi.org/10.12688/f1000research.145146.2) First published: 03 Jan 2024, 13:1 (https://doi.org/10.12688/f1000research.145146.1) Latest published: 09 Jul 2024, 13:1 (https://doi.org/10.12688/f1000research.145146.2)

Revised Amendments from Version 1

In this updated version of the article, Table 2 has been moved to be presented before Figure 1, as to show the information regarding the antibodies prior to the results. As well, we have included an additional limitation to our protocol in the results and discussion section.

See the authors' detailed response to the review by Dario Siniscalco and Mauro Finicelli
See the authors' detailed response to the review by Nadia Bouhamdani

Introduction

Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that regulates various cellular functions including cell metabolism, cell cycle, DNA replication, transcription and translation, signal transduction, cell mobility and apoptosis.¹^–⁴ PP2A has a heterotrimeric form consisting of 3 subunits: catalytic, scaffold and regulatory.⁵ The specificity and activity of the PP2A enzyme is regulated by the associated regulatory subunit, which controls substrate selectivity and activity of PP2A.⁶^–⁹

The PP2A-B56 family of the regulatory subunit has 5 isoforms; α, β, γ, δ, and ε. The PPP2R5D gene encodes serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D).⁹^,¹⁰ This δ isoform is highly expressed in neuronal tissues and is crucial for cell cycle regulation. It controls the ability of PP2A to negatively regulate PI3K/AKT signalling pathways and modulates glycogen synthase kinase-3 β and cyclin-dependent kinase 5 activities by controlling tau phosphorylation.⁸^,¹¹ Missense mutations in PPP2R5D have been linked to neurodevelopmental disorders such as neurodevelopmental delay,¹² intellectual disability¹³^–¹⁵ and autism spectrum disorder.¹⁶^–²⁰ The majority of these mutations lead to Jordan’s syndrome, an autosomal dominant illness associated with intellectual disabilities and autism spectrum disorders.²⁰ Exploring the intricate network involving PPP2R5D as it impacts cell cycle regulation in neuronal processes could significantly enhance the potential for uncovering novel treatments for neurodevelopmental disorders. Further research is required and would be facilitated with the availability of high-quality antibodies.

This research is part of a broader collaborative initiative in which academics, funders and commercial antibody manufacturers are working together to address antibody reproducibility issues by characterizing commercial antibodies for human proteins using standardized protocols, and openly sharing the data.²¹^–²³ Here, we evaluated the performance of six commercially-available antibodies for PPP2R5D in Western Blot, immunoprecipitation and immunofluorescence using a knockout based approach. This article serves as a valuable guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of PPP2R5D properties and function.

Results and discussion

Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells.²⁴^,²⁵ The first step is to identify a cell line(s) that expresses sufficient endogenous levels of a given protein to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log₂ (transcripts per million “TPM” + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the PPP2R5D transcript at RNA levels above the average range of cancer cells analyzed. Parental and PPP2R5D KO HAP1 cells were obtained from Horizon Discovery (Table 1).

Table 1. Summary of the cell lines used.

Institution	Catalog number	RRID (Cellosaurus)	Cell line	Genotype
Horizon Discovery	C631	CVCL_Y019	HAP1	WT
Horizon Discovery	HZGHC003155c003	CVCL_TG11	HAP1	PPP2R5D KO

For Western Blot experiments, we resolved proteins from WT and PPP2R5D KO cell extracts and probed them side-by-side with all antibodies in parallel (Table 2, Figure 1).

Table 2. Summary of the PPP2R5D antibodies tested.

Company	Catalog number	Lot number	RRID (Antibody Registry)	Clonality	Clone ID	Host	Concentration (μg/μL)	Vendors recommended applications
Abcam	ab188323**	1023484-1	AB_3065198	recombinant- mono	EPR15617	rabbit	0.239	WB, IP, IF
Abcam	ab188325**	GR169019-2	AB_3065199	recombinant- mono	EPR15617-50	rabbit	0.315	WB, IP
Cell Signaling Technology	5687*	1	AB_10829041	monoclonal	H5D12	mouse	0.077	WB, IP
GeneTex	GTX106401	39694	AB_1241205	polyclonal	-	rabbit	1.0	WB
Thermo Fisher Scientific	MA5-18066*	YE3914232	AB_2539449	monoclonal	H5D12	mouse	1.0	WB, IP
Thermo Fisher Scientific	MA5-26647*	YE3914196	AB_2725096	monoclonal	OTI3B3	mouse	1.0	WB

* Monoclonal antibody.

** Recombinant antibody.

Figure 1. PPP2R5D antibody screening by Western Blot.

A) Lysates of HAP1 WT and PPP2R5D KO were prepared in RIPA buffer, and 30 μg of protein were processed for Western Blot with the indicated PPP2R5D antibodies. The Ponceau stained transfers of each blot are shown. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 5687*, MA5-18066* and MA5-26647*, which were titrated to the corresponding dilutions found below, as the signals were too weak when following the supplier’s recommendations. Antibody dilution used: ab188323** at 1/10 000, ab188325** at 1/10 000, 5687* at 1/200, GTX106401 at 1/500, MA5-18066* at 1/200, MA5-26647* at 1/200. B) The observed molecular weight of PPP2R5D varies according to the lysis buffer used. HAP1 WT and PPP2R5D KO lysates were prepared in RIPA or IP lysis buffer and the molecular weight of PPP2R5D was assessed by WB. The composition of lysis buffers is indicated in the Methods section. PPP2R5D ran primarily at ~65 kDa or ~75 kDa when lysates were prepared with RIPA or IP lysis buffer, respectively. ab188323** was used at 1/10 000 for Western Blot.

As per our standard procedure, we next used the antibodies to immunoprecipite PPP2R5D from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the PPP2R5D protein in extracts, in the immunodepleted extracts and in the immunoprecipitates (Figure 2).

Figure 2. PPP2R5D antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and immunoprecipitation was performed using 2.0 μg of the indicated PPP2R5D antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for Western Blot with the indicated PPP2R5D antibody. For Western Blot, ab188323** was used at 1/5000. The Ponceau stained transfers of each blot are shown. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, antibodies were screened using a mosaic strategy, as per our standard procedure. First, HAP1 WT and PPP2R5D KO cell lines were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the six PPP2R5D antibodies were evaluated. Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3). Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested. The images presented in Figure 3 are representative of the results of this analysis.

Figure 3. PPP2R5D antibody screening by immunofluorescence.

HAP1 WT and PPP2R5D KO cells were labelled with a green or a far-red fluorescent dye, respectively. WT and KO cells were mixed and plated to a 1:1 ratio in a 96-well plate with optically clear flat-bottom. Cells were stained with the indicated PPP2R5D antibodies (first panel) and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI (second panel). Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT cells were outlined with green dashed lines while KO cells were outlined with magenta dashed lines. When the concentration was not indicated by the supplier, which was the case for antibodies ab188325**, 5687*, GTX106401, MA5-18066* and MA5-26647*, we tested antibodies at the concentrations found below. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab188323** at 1/200, ab188325** at 1/300, 5687* at 1/70, GTX106401 at 1/1000, MA5-18066* at 1/1000, MA5-26647* at 1/1000. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In summary, we have screened six PPP2R5D commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence. Several high-quality antibodies that successfully detect PPP2R5D under our standardized experimental conditions were identified. In our effort to address the antibody reliability and reproducibility challenges in scientific research, the authors recommend the antibodies that demonstrated to be underperforming under our standard procedure be removed from the commercial antibody market. However, the authors do not engage in result analysis or offer explicit antibody recommendations. A limitation of this study is the use of universal protocols - any conclusions remain relevant within the confines of the experimental setup and cell line used in this study. Another limitation is the orthogonal strategy which evaluates the antibodies in one cancer cell line, rather than various cell lines expressing mutants or variants of the target protein. Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles. This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. Guidelines on how to interpret the antibody characterization data in this study are available on the YCharOS Gateway.²⁶

The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports.²⁷^,²⁸

Methods

Antibodies

All PPP2R5D antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly.²⁹ Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 65-6120 and 62-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).

Cell culture

Both HAP1 WT and PPP2R5D KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly.³⁰ Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (Wisent, cat. number 080450), 2 mM L-glutamate (Wisent cat. number 609065, 100 IU penicillin and 100 μg/mL streptomycin (Wisent cat. number 450201).

Antibody screening by Western Blot

HAP1 WT and PPP2R5D KO were collected RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat. number 89901) supplemented with 1× protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and Western Blot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.

Western Blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels from Thermo Fisher Scientific (cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer from bio-Rad (cat. number 1610772), loaded in Laemmli loading sample buffer from Thermo Fisher Scientific (cat. number AAJ61337AD) and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat. number BP103-10) which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% milk in TBS with 0.1% Tween 20 (TBST) (Cell Signalling Technology, cat. number 9997). Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST. Membranes were developed with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) Or Clarity Western ECL Substrate from Bio-Rad (cat. number 1705061) prior to detection with the iBright™ CL1500 Imaging System from Thermo Fisher Scientific (cat. number A44240).

Antibody screening by immunoprecipitation

Antibody-bead conjugates were prepared by adding 2 μg of antibody at an unknown concentration to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30 μL of Dynabeads protein A - (for rabbit antibodies) or protein G - (for mouse) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.

HAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) supplemented with protease inhibitor. Lysates were rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western Blot on a precast midi 4-20% Tris-Glycine polyacrylamide gels. VeriBlot for IP Detection Reagent:HRP (Abcam, cat. number ab131366) was used as a secondary detection system at a concentration of 0.1 μg/mL.

Antibody screening by immunofluorescence

HAP1 WT and PPP2R5D KO were labelled with a green and a far-red fluorescence dye, respectively. The fluorescent dyes used are from Thermo Fisher Scientific (cat. number C2925 and C34565). The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat. Number D3571) fluorescent stain. WT and KO cells were then plated in a 96-well plate with optically clear flat-bottom as a mosaic and incubated for 24 hrs in a cell culture incubator at 37^oC, 5% CO₂. Cells were fixed in 4% paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10 ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL). Cells were permeabilized in PBS with 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature and blocked with PBS with 5% bovine serum albumin (BSA) (Wisent, cat. number 800-095), 5% goat serum (Gibco, cat. number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature. Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary PPP2R5D antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI. Cells were washed 3 × 10 min with IF buffer and once with PBS.

Images were acquired on an ImageXpress micro widefield high-content microscopy system (Molecular Devices), using a 20× NA 0.95 water objective lens and scientific CMOS camera (16-bit, 1.97 mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTracker^TM Green, Alexa fluor 555 and CellTracker^TM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTracker^TM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns.³¹ Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Photoshop (version 24.1.2) to adjust contrast then assembled with Adobe Illustrator (version 27.3.1).

Data availability

Underlying data

Zenodo: Antibody Characterization Report for PPP2R5D, https://doi.org/10.5281/zenodo.10108248.²⁷

Zenodo: Dataset for the PPP2R5D antibody screening study, https://doi.org/10.5281/zenodo.10119578.²⁸

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgment

We would like to thank the NeuroSGC/YCharOS/EDDU collaborative group for their important contribution to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources and collaboration. We would also like to thank the Advanced BioImaging Facility (ABIF) consortium for their image analysis pipeline development and conduction (RRID:SCR_017697). Members of each group can be found below.

NeuroSGC/YCharOS/EDDU collaborative group: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.

ABIF consortium: Claire M. Brown and Joel Ryan.

Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no. OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no. 875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.10108248).

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Version 2

VERSION 2 PUBLISHED 03 Jan 2024

Author details Author details

¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada

Riham Ayoubi
Roles: Investigation, Methodology, Validation

Maryam Fotouhi
Roles: Investigation, Visualization

Charles Alende
Roles: Investigation, Visualization

Kathleen Southern
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Carl Laflamme
Roles: Conceptualization, Data Curation, Funding Acquisition, Methodology, Resources, Supervision, Validation

Competing interests

For this project, the laboratory of Peter McPherson developed partnerships with high-quality antibody manufacturers and knockout cell line providers. The partners provide antibodies and knockout cell lines to the McPherson laboratory at no cost. These partners include: - Abcam-Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -GeneTex – Horizon Discovery – Proteintech – Synaptic Systems –Thermo Fisher Scientific.

Grant information

The grant was from a Canadian Institutes of Health Research Foundation (grant no. FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (OGI-210). The study was also funded in part by the Simons Foundation Autism Research Initiative (SFARI). RA is supported by a Mitacs fellowship. 
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Ayoubi R, Fotouhi M, Alende C et al. A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence [version 2; peer review: 2 approved]. F1000Research 2024, 13:1 (https://doi.org/10.12688/f1000research.145146.2)

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Reviewer Report 07 Aug 2024

Nadia Bouhamdani, Medical Genetics Department & Department of Chemistry and Biochemistry, Vitalité Health Network, Dr Georges-L.-Dumont University Hospital Center, Université de Sherbrooke, Université de Moncton, Moncton, New Brunswick, Canada

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https://doi.org/10.5256/f1000research.168890.r300981

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Reviewer Report 30 Jul 2024

Dario Siniscalco, University of Campania, Naples, Italy

Mauro Finicelli, National Research Council of Italy, Naples, Italy

Approved

https://doi.org/10.5256/f1000research.168890.r303543

- It would be interesting to see also other cell lines (i.e. nervous system derived cells or tissues).
- It would be more informative if the authors had used 4 cell lines for their experiments. This is to say, the use of a highly-expressing PPP2R5D cell line, a normal-expressing PPP2R5D cell line, a low-expressing cell line and a KO cell line. The combination of these results could give a valid overview about the effectiveness of the antibodies tested in all the assays.
- Although the data from DepMap transcriptomics database about the expression levels of PPP2R5D in HAP1 cells and KO ones, it could be useful a check of the authors in their cell lines. The authors should test the effective levels on PPP2R5D mRNA levels in both the cell lines used for their experiments.
- One of the ab used has been discontinued by the company. Authors should consider this in discussion section.
- It could be useful to add a direct link to the ab's vendor webpage in Table 2, as the antibody registry.org only has a link to the main vendor's webpage and not to the specific ab's webpage.
- Methods, WB: two different ECL systems have been employed. This could affect signal detection. Briefly explain.
- ICC: the authors must indicate the rationale used for identification of the antibody dilution applied in ICC experiments. E.g., although for MA5-18066 the antibody dilution recommended in the brochure is assay-dependent in HeLa cells the antibody was diluted 1:100 with a good nuclear signal; why did the authors used 1:1000? Moreover, it is not clearly reported if the authors used a negative control (i.e., reaction without primary antibody) for each experiment.
- About ICC experiments, the authors should provide a single image for PPP2R5D expression levels in HAP1 and KO cells, respectively, to corroborate the results obtained in co-cultured cells. I appreciate if the authors could provide the single channel and merged images representative of the experiments.

Is the rationale for creating the dataset(s) clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of methods and materials provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: autism; nervous system; gene expression; protein levels; enzyme activity; cell biology; miRNA.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response 13 Dec 2024

Carl Laflamme, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Dec 2024

Author Response

Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the ... Continue reading Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the PPP2R5D protein itself. The primary objective of this work is to provide an unbiased evaluation of the performance of currently available PPP2R5D antibodies. The processes and methodologies employed in this study were endorsed by 12 antibody manufacturers and disease foundations. Additionally, the protocols have been shared as a preprint and are accepted for publication in Nature Protocols, with an expected release by the end of 2024.

As demonstrated by DepMap, which provides RNA sequencing data for thousands of cancer cell lines, PPP2R5D is ubiquitously expressed. The antibody ab188323 has been previously validated in western blot analysis by Abcam using various cell lines, including A431, HeLa, and MCF7, showing a similar pattern to our findings in HAP1 cells. We acknowledge that RNA and protein levels may not always correlate perfectly. However, we confirm that HAP1 expresses adequate PPP2R5D protein levels as a subset of antibodies successfully detected PPP2R5D, as evidenced by the lack of signal in knockout (KO) lysates.

The polyclonal antibody GTX106401 has been discontinued, which is a common occurrence with polyclonal products due to their finite nature. This underscores the importance of the YCharOS initiative and its industry partners focusing on recombinant and monoclonal antibodies. Of the six antibodies tested in this study, five fall into these categories.

Regarding your specific concerns:

1) Antibody detection sensitivity:
Our standard protocol begins with the Pierce ECL system using the manufacturer-recommended antibody dilutions. For antibodies yielding weak signals, we increase the concentration fivefold and switch to the Bio-Rad ECL enhanced system, which has provided stronger signals in our experience.

2) ICC specificity testing:
In ICC experiments, antibodies were evaluated using a mosaic of wild-type (WT) and KO cell lines to assess specificity. For example, MA5-18066 showed similar staining in both WT (green outline) and KO (pink outline) cell lines, indicating it is unsuitable under the tested conditions. However, two antibodies demonstrated specific cytoplasmic staining, clearly observed by the lack of signal in KO cells, with no nuclear signal.

Testing antibody performance in WT and KO cell lines within the same field of view represents the gold standard in antibody characterization. This approach minimizes user and imaging biases and ensures that identical microscopy parameters are applied to both conditions, enhancing the reliability of the results.

3) Links from the Antibody Registry to vendor websites:
I contacted the Antibody Registry to address your concern, and they confirmed that they maintain updated links to the direct product pages for all companies that provide them. Unfortunately, not all companies supply these updates. The current Table 2 provides all the necessary information to trace the antibodies used in this study.

We hope this addresses your feedback comprehensively.

Thank you once again for your valuable input.

Best regards,
Carl Laflamme
Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the PPP2R5D protein itself. The primary objective of this work is to provide an unbiased evaluation of the performance of currently available PPP2R5D antibodies. The processes and methodologies employed in this study were endorsed by 12 antibody manufacturers and disease foundations. Additionally, the protocols have been shared as a preprint and are accepted for publication in Nature Protocols, with an expected release by the end of 2024.

As demonstrated by DepMap, which provides RNA sequencing data for thousands of cancer cell lines, PPP2R5D is ubiquitously expressed. The antibody ab188323 has been previously validated in western blot analysis by Abcam using various cell lines, including A431, HeLa, and MCF7, showing a similar pattern to our findings in HAP1 cells. We acknowledge that RNA and protein levels may not always correlate perfectly. However, we confirm that HAP1 expresses adequate PPP2R5D protein levels as a subset of antibodies successfully detected PPP2R5D, as evidenced by the lack of signal in knockout (KO) lysates.

The polyclonal antibody GTX106401 has been discontinued, which is a common occurrence with polyclonal products due to their finite nature. This underscores the importance of the YCharOS initiative and its industry partners focusing on recombinant and monoclonal antibodies. Of the six antibodies tested in this study, five fall into these categories.

Regarding your specific concerns:

1) Antibody detection sensitivity:
Our standard protocol begins with the Pierce ECL system using the manufacturer-recommended antibody dilutions. For antibodies yielding weak signals, we increase the concentration fivefold and switch to the Bio-Rad ECL enhanced system, which has provided stronger signals in our experience.

2) ICC specificity testing:
In ICC experiments, antibodies were evaluated using a mosaic of wild-type (WT) and KO cell lines to assess specificity. For example, MA5-18066 showed similar staining in both WT (green outline) and KO (pink outline) cell lines, indicating it is unsuitable under the tested conditions. However, two antibodies demonstrated specific cytoplasmic staining, clearly observed by the lack of signal in KO cells, with no nuclear signal.

Testing antibody performance in WT and KO cell lines within the same field of view represents the gold standard in antibody characterization. This approach minimizes user and imaging biases and ensures that identical microscopy parameters are applied to both conditions, enhancing the reliability of the results.

3) Links from the Antibody Registry to vendor websites:
I contacted the Antibody Registry to address your concern, and they confirmed that they maintain updated links to the direct product pages for all companies that provide them. Unfortunately, not all companies supply these updates. The current Table 2 provides all the necessary information to trace the antibodies used in this study.

We hope this addresses your feedback comprehensively.

Thank you once again for your valuable input.

Best regards,
Carl Laflamme
Competing Interests: No competing interests were disclosed. Close
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Reviewer Response 28 Dec 2024

Dario Siniscalco, University of Campania, Naples, Italy

28 Dec 2024

Reviewer Response

Authors succesfully answered to my comments.
Competing Interests: No competing interests were disclosed.
Authors succesfully answered to my comments.
Authors succesfully answered to my comments.
Competing Interests: No competing interests were disclosed. Close
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COMMENTS ON THIS REPORT

Author Response 13 Dec 2024

Carl Laflamme, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

13 Dec 2024

Author Response

Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the ... Continue reading Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the PPP2R5D protein itself. The primary objective of this work is to provide an unbiased evaluation of the performance of currently available PPP2R5D antibodies. The processes and methodologies employed in this study were endorsed by 12 antibody manufacturers and disease foundations. Additionally, the protocols have been shared as a preprint and are accepted for publication in Nature Protocols, with an expected release by the end of 2024.

As demonstrated by DepMap, which provides RNA sequencing data for thousands of cancer cell lines, PPP2R5D is ubiquitously expressed. The antibody ab188323 has been previously validated in western blot analysis by Abcam using various cell lines, including A431, HeLa, and MCF7, showing a similar pattern to our findings in HAP1 cells. We acknowledge that RNA and protein levels may not always correlate perfectly. However, we confirm that HAP1 expresses adequate PPP2R5D protein levels as a subset of antibodies successfully detected PPP2R5D, as evidenced by the lack of signal in knockout (KO) lysates.

The polyclonal antibody GTX106401 has been discontinued, which is a common occurrence with polyclonal products due to their finite nature. This underscores the importance of the YCharOS initiative and its industry partners focusing on recombinant and monoclonal antibodies. Of the six antibodies tested in this study, five fall into these categories.

Regarding your specific concerns:

1) Antibody detection sensitivity:
Our standard protocol begins with the Pierce ECL system using the manufacturer-recommended antibody dilutions. For antibodies yielding weak signals, we increase the concentration fivefold and switch to the Bio-Rad ECL enhanced system, which has provided stronger signals in our experience.

2) ICC specificity testing:
In ICC experiments, antibodies were evaluated using a mosaic of wild-type (WT) and KO cell lines to assess specificity. For example, MA5-18066 showed similar staining in both WT (green outline) and KO (pink outline) cell lines, indicating it is unsuitable under the tested conditions. However, two antibodies demonstrated specific cytoplasmic staining, clearly observed by the lack of signal in KO cells, with no nuclear signal.

Testing antibody performance in WT and KO cell lines within the same field of view represents the gold standard in antibody characterization. This approach minimizes user and imaging biases and ensures that identical microscopy parameters are applied to both conditions, enhancing the reliability of the results.

3) Links from the Antibody Registry to vendor websites:
I contacted the Antibody Registry to address your concern, and they confirmed that they maintain updated links to the direct product pages for all companies that provide them. Unfortunately, not all companies supply these updates. The current Table 2 provides all the necessary information to trace the antibodies used in this study.

We hope this addresses your feedback comprehensively.

Thank you once again for your valuable input.

Best regards,
Carl Laflamme
Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the PPP2R5D protein itself. The primary objective of this work is to provide an unbiased evaluation of the performance of currently available PPP2R5D antibodies. The processes and methodologies employed in this study were endorsed by 12 antibody manufacturers and disease foundations. Additionally, the protocols have been shared as a preprint and are accepted for publication in Nature Protocols, with an expected release by the end of 2024.

As demonstrated by DepMap, which provides RNA sequencing data for thousands of cancer cell lines, PPP2R5D is ubiquitously expressed. The antibody ab188323 has been previously validated in western blot analysis by Abcam using various cell lines, including A431, HeLa, and MCF7, showing a similar pattern to our findings in HAP1 cells. We acknowledge that RNA and protein levels may not always correlate perfectly. However, we confirm that HAP1 expresses adequate PPP2R5D protein levels as a subset of antibodies successfully detected PPP2R5D, as evidenced by the lack of signal in knockout (KO) lysates.

The polyclonal antibody GTX106401 has been discontinued, which is a common occurrence with polyclonal products due to their finite nature. This underscores the importance of the YCharOS initiative and its industry partners focusing on recombinant and monoclonal antibodies. Of the six antibodies tested in this study, five fall into these categories.

Regarding your specific concerns:

1) Antibody detection sensitivity:
Our standard protocol begins with the Pierce ECL system using the manufacturer-recommended antibody dilutions. For antibodies yielding weak signals, we increase the concentration fivefold and switch to the Bio-Rad ECL enhanced system, which has provided stronger signals in our experience.

2) ICC specificity testing:
In ICC experiments, antibodies were evaluated using a mosaic of wild-type (WT) and KO cell lines to assess specificity. For example, MA5-18066 showed similar staining in both WT (green outline) and KO (pink outline) cell lines, indicating it is unsuitable under the tested conditions. However, two antibodies demonstrated specific cytoplasmic staining, clearly observed by the lack of signal in KO cells, with no nuclear signal.

Testing antibody performance in WT and KO cell lines within the same field of view represents the gold standard in antibody characterization. This approach minimizes user and imaging biases and ensures that identical microscopy parameters are applied to both conditions, enhancing the reliability of the results.

3) Links from the Antibody Registry to vendor websites:
I contacted the Antibody Registry to address your concern, and they confirmed that they maintain updated links to the direct product pages for all companies that provide them. Unfortunately, not all companies supply these updates. The current Table 2 provides all the necessary information to trace the antibodies used in this study.

We hope this addresses your feedback comprehensively.

Thank you once again for your valuable input.

Best regards,
Carl Laflamme
Competing Interests: No competing interests were disclosed. Close
Report a concern
Reviewer Response 28 Dec 2024

Dario Siniscalco, University of Campania, Naples, Italy

28 Dec 2024

Reviewer Response

Authors succesfully answered to my comments.
Competing Interests: No competing interests were disclosed.
Authors succesfully answered to my comments.
Authors succesfully answered to my comments.
Competing Interests: No competing interests were disclosed. Close
Report a concern

Version 1

VERSION 1

PUBLISHED 03 Jan 2024

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Reviewer Report 20 May 2024

Approved with Reservations

https://doi.org/10.5256/f1000research.159050.r269488

The authors characterize six commercially available antibodies for PPP2R5D using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. The main objective of this study is to provide a published report to guide researchers in the field in selecting the most appropriate available antibodies for their specific research needs.

In general, this data note article is clear and straight forward; however, important details are lacking. Here are some comments/questions that need to be addressed:

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.

Is the rationale for creating the dataset(s) clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of methods and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Cancer research, proteomics, functional genomics, rare disease research.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Author Response 09 Jul 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

09 Jul 2024

Author Response
Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for ... Continue reading
Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence”. We appreciate your feedback and commitment to enhancing the reproducibility and comprehension of the antibody characterization data provided in the article. Please see below our responses to your comments/questions. A new version of the article will be submitted shortly after to address a few of your concerns.

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Response: From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 5.7 log₂ (TPM+1), which is significantly above the minimum threshold level we’ve established. From the results, it is evident that the selection of HAP1 as cell line for antibody testing was appropriate, as PPP2R5D was easily detected in the lysates when testing the antibodies by western blot. We acknowledge that antibody characterization data generated by following the described protocols does not provide a comprehensive evaluation of the tested antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using KO cell lines, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Nature Protocols (DOI:10.21203/rs.3.pex-2607/v1).

2. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
3. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
Response to point 2 & 3: We understand that mutations in the epitope coding sequence or elsewhere in the gene coding for the intended target could affect antibody binding, although we think this would be a rare occurrence. However, this is an inherent limitation of any approach that uses cancer cell lines. We have addressed this limitation in the summary paragraph of the results and discussion section.

4. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
Response: The antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who have access to 80% of renewable antibodies covered in commercial catalogs, the other 20% of antibodies from sources that are not partners, why is why we don’t test them. When the partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

5. An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
Response: As indicated on their catalog pages, these antibodies are intended to target PPP2R5D, which is why we have tested them, side-by-side, using a PPP2R5D KO cell line. This allows viewers to determine whether the antibodies are detecting PPP2R5D or identify certain variants which they can then use to further validate for cross-reactivity with other PP2A-B56 isoforms. Unfortunately, this degree of validation is outside the scope of our study but we encourage experts in the field to utilize the validation data provided as a starting point to enhance their PPP2R5D related research.

6. I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
Response: Thank you for bringing up this concern. The newly submitted version of this article will present Table 2 before presenting the results.

7. In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
Response: When carrying out the IP experiment (Figure 2), the lysates were freshly prepared and antibodies were immediately processed for IP, which was not the case for the lysis buffer comparison experiment (Figure 1B). In Figure 1, frozen lysates were used to carry out the experiments (A and B). This can explain the difference in signal pattern seen in Figure 2 vs Figure 1B.

8. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.
Response: As described in the final paragraphs of the results and discussion section, the authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1).
Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded.
Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence”. We appreciate your feedback and commitment to enhancing the reproducibility and comprehension of the antibody characterization data provided in the article. Please see below our responses to your comments/questions. A new version of the article will be submitted shortly after to address a few of your concerns.

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Response: From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 5.7 log₂ (TPM+1), which is significantly above the minimum threshold level we’ve established. From the results, it is evident that the selection of HAP1 as cell line for antibody testing was appropriate, as PPP2R5D was easily detected in the lysates when testing the antibodies by western blot. We acknowledge that antibody characterization data generated by following the described protocols does not provide a comprehensive evaluation of the tested antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using KO cell lines, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Nature Protocols (DOI:10.21203/rs.3.pex-2607/v1).

2. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
3. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
Response to point 2 & 3: We understand that mutations in the epitope coding sequence or elsewhere in the gene coding for the intended target could affect antibody binding, although we think this would be a rare occurrence. However, this is an inherent limitation of any approach that uses cancer cell lines. We have addressed this limitation in the summary paragraph of the results and discussion section.

4. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
Response: The antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who have access to 80% of renewable antibodies covered in commercial catalogs, the other 20% of antibodies from sources that are not partners, why is why we don’t test them. When the partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

5. An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
Response: As indicated on their catalog pages, these antibodies are intended to target PPP2R5D, which is why we have tested them, side-by-side, using a PPP2R5D KO cell line. This allows viewers to determine whether the antibodies are detecting PPP2R5D or identify certain variants which they can then use to further validate for cross-reactivity with other PP2A-B56 isoforms. Unfortunately, this degree of validation is outside the scope of our study but we encourage experts in the field to utilize the validation data provided as a starting point to enhance their PPP2R5D related research.

6. I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
Response: Thank you for bringing up this concern. The newly submitted version of this article will present Table 2 before presenting the results.

7. In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
Response: When carrying out the IP experiment (Figure 2), the lysates were freshly prepared and antibodies were immediately processed for IP, which was not the case for the lysis buffer comparison experiment (Figure 1B). In Figure 1, frozen lysates were used to carry out the experiments (A and B). This can explain the difference in signal pattern seen in Figure 2 vs Figure 1B.

8. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.
Response: As described in the final paragraphs of the results and discussion section, the authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1).
Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded.
Competing Interests: No competing interests were disclosed. Close
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Author Response 09 Jul 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

09 Jul 2024

Author Response
Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for ... Continue reading
Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence”. We appreciate your feedback and commitment to enhancing the reproducibility and comprehension of the antibody characterization data provided in the article. Please see below our responses to your comments/questions. A new version of the article will be submitted shortly after to address a few of your concerns.

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Response: From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 5.7 log₂ (TPM+1), which is significantly above the minimum threshold level we’ve established. From the results, it is evident that the selection of HAP1 as cell line for antibody testing was appropriate, as PPP2R5D was easily detected in the lysates when testing the antibodies by western blot. We acknowledge that antibody characterization data generated by following the described protocols does not provide a comprehensive evaluation of the tested antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using KO cell lines, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Nature Protocols (DOI:10.21203/rs.3.pex-2607/v1).

2. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
3. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
Response to point 2 & 3: We understand that mutations in the epitope coding sequence or elsewhere in the gene coding for the intended target could affect antibody binding, although we think this would be a rare occurrence. However, this is an inherent limitation of any approach that uses cancer cell lines. We have addressed this limitation in the summary paragraph of the results and discussion section.

4. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
Response: The antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who have access to 80% of renewable antibodies covered in commercial catalogs, the other 20% of antibodies from sources that are not partners, why is why we don’t test them. When the partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

5. An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
Response: As indicated on their catalog pages, these antibodies are intended to target PPP2R5D, which is why we have tested them, side-by-side, using a PPP2R5D KO cell line. This allows viewers to determine whether the antibodies are detecting PPP2R5D or identify certain variants which they can then use to further validate for cross-reactivity with other PP2A-B56 isoforms. Unfortunately, this degree of validation is outside the scope of our study but we encourage experts in the field to utilize the validation data provided as a starting point to enhance their PPP2R5D related research.

6. I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
Response: Thank you for bringing up this concern. The newly submitted version of this article will present Table 2 before presenting the results.

7. In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
Response: When carrying out the IP experiment (Figure 2), the lysates were freshly prepared and antibodies were immediately processed for IP, which was not the case for the lysis buffer comparison experiment (Figure 1B). In Figure 1, frozen lysates were used to carry out the experiments (A and B). This can explain the difference in signal pattern seen in Figure 2 vs Figure 1B.

8. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.
Response: As described in the final paragraphs of the results and discussion section, the authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1).
Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded.
Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence”. We appreciate your feedback and commitment to enhancing the reproducibility and comprehension of the antibody characterization data provided in the article. Please see below our responses to your comments/questions. A new version of the article will be submitted shortly after to address a few of your concerns.

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Response: From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 5.7 log₂ (TPM+1), which is significantly above the minimum threshold level we’ve established. From the results, it is evident that the selection of HAP1 as cell line for antibody testing was appropriate, as PPP2R5D was easily detected in the lysates when testing the antibodies by western blot. We acknowledge that antibody characterization data generated by following the described protocols does not provide a comprehensive evaluation of the tested antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using KO cell lines, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Nature Protocols (DOI:10.21203/rs.3.pex-2607/v1).

2. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
3. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
Response to point 2 & 3: We understand that mutations in the epitope coding sequence or elsewhere in the gene coding for the intended target could affect antibody binding, although we think this would be a rare occurrence. However, this is an inherent limitation of any approach that uses cancer cell lines. We have addressed this limitation in the summary paragraph of the results and discussion section.

4. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
Response: The antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who have access to 80% of renewable antibodies covered in commercial catalogs, the other 20% of antibodies from sources that are not partners, why is why we don’t test them. When the partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

5. An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
Response: As indicated on their catalog pages, these antibodies are intended to target PPP2R5D, which is why we have tested them, side-by-side, using a PPP2R5D KO cell line. This allows viewers to determine whether the antibodies are detecting PPP2R5D or identify certain variants which they can then use to further validate for cross-reactivity with other PP2A-B56 isoforms. Unfortunately, this degree of validation is outside the scope of our study but we encourage experts in the field to utilize the validation data provided as a starting point to enhance their PPP2R5D related research.

6. I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
Response: Thank you for bringing up this concern. The newly submitted version of this article will present Table 2 before presenting the results.

7. In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
Response: When carrying out the IP experiment (Figure 2), the lysates were freshly prepared and antibodies were immediately processed for IP, which was not the case for the lysis buffer comparison experiment (Figure 1B). In Figure 1, frozen lysates were used to carry out the experiments (A and B). This can explain the difference in signal pattern seen in Figure 2 vs Figure 1B.

8. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.
Response: As described in the final paragraphs of the results and discussion section, the authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1).
Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded.
Competing Interests: No competing interests were disclosed. Close
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Version 2

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Nadia Bouhamdani, Vitalité Health Network, Dr Georges-L.-Dumont University Hospital Center, Université de Sherbrooke, Université de Moncton, Moncton, Canada
Dario Siniscalco, University of Campania, Naples, Italy

Mauro Finicelli, National Research Council of Italy, Naples, Italy

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07 Aug 2024 | for Version 2

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Approved

The revisions by the authors are sufficient.

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Cancer research, proteomics, functional genomics, rare disease research.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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30 Jul 2024 | for Version 2

Dario Siniscalco, University of Campania, Naples, Italy

Mauro Finicelli, National Research Council of Italy, Naples, Italy

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Approved

Is the rationale for creating the dataset(s) clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Yes
Are sufficient details of methods and materials provided to allow replication by others?

Partly
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

autism; nervous system; gene expression; protein levels; enzyme activity; cell biology; miRNA.

We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Author Response

13 Dec 2024

Carl Laflamme, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

Dear Dr. Siniscalco,

Thank you for your feedback on the PPP2R5D antibody characterization data note.

The authors of this study are antibody experts, though not specialists on the PPP2R5D protein itself. The primary objective of this work is to provide an unbiased evaluation of the performance of currently available PPP2R5D antibodies. The processes and methodologies employed in this study were endorsed by 12 antibody manufacturers and disease foundations. Additionally, the protocols have been shared as a preprint and are accepted for publication in Nature Protocols, with an expected release by the end of 2024.

As demonstrated by DepMap, which provides RNA sequencing data for thousands of cancer cell lines, PPP2R5D is ubiquitously expressed. The antibody ab188323 has been previously validated in western blot analysis by Abcam using various cell lines, including A431, HeLa, and MCF7, showing a similar pattern to our findings in HAP1 cells. We acknowledge that RNA and protein levels may not always correlate perfectly. However, we confirm that HAP1 expresses adequate PPP2R5D protein levels as a subset of antibodies successfully detected PPP2R5D, as evidenced by the lack of signal in knockout (KO) lysates.

The polyclonal antibody GTX106401 has been discontinued, which is a common occurrence with polyclonal products due to their finite nature. This underscores the importance of the YCharOS initiative and its industry partners focusing on recombinant and monoclonal antibodies. Of the six antibodies tested in this study, five fall into these categories.

Regarding your specific concerns:

1) Antibody detection sensitivity:
Our standard protocol begins with the Pierce ECL system using the manufacturer-recommended antibody dilutions. For antibodies yielding weak signals, we increase the concentration fivefold and switch to the Bio-Rad ECL enhanced system, which has provided stronger signals in our experience.

2) ICC specificity testing:
In ICC experiments, antibodies were evaluated using a mosaic of wild-type (WT) and KO cell lines to assess specificity. For example, MA5-18066 showed similar staining in both WT (green outline) and KO (pink outline) cell lines, indicating it is unsuitable under the tested conditions. However, two antibodies demonstrated specific cytoplasmic staining, clearly observed by the lack of signal in KO cells, with no nuclear signal.

Testing antibody performance in WT and KO cell lines within the same field of view represents the gold standard in antibody characterization. This approach minimizes user and imaging biases and ensures that identical microscopy parameters are applied to both conditions, enhancing the reliability of the results.

3) Links from the Antibody Registry to vendor websites:
I contacted the Antibody Registry to address your concern, and they confirmed that they maintain updated links to the direct product pages for all companies that provide them. Unfortunately, not all companies supply these updates. The current Table 2 provides all the necessary information to trace the antibodies used in this study.

We hope this addresses your feedback comprehensively.

Thank you once again for your valuable input.

Best regards,
Carl Laflamme

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No competing interests were disclosed.

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20 May 2024 | for Version 1

20 Views Cite this report Responses(1)

Approved With Reservations

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.

Is the rationale for creating the dataset(s) clearly described?

Yes
Are the protocols appropriate and is the work technically sound?

Partly
Are sufficient details of methods and materials provided to allow replication by others?

Yes
Are the datasets clearly presented in a useable and accessible format?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Cancer research, proteomics, functional genomics, rare disease research.

Respond to this report

Responses (1)

Author Response

09 Jul 2024

Kathleen Southern, Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, H3A 2B4, Canada

Dear Nadia Bouhamdani, thank you for your comprehensive review of the article “A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence”. We appreciate your feedback and commitment to enhancing the reproducibility and comprehension of the antibody characterization data provided in the article. Please see below our responses to your comments/questions. A new version of the article will be submitted shortly after to address a few of your concerns.

In the section ‘results and discussion’, the authors mention that cell models used to characterize PPP2R5D antibodies were chosen based on analyzing ‘DepMap transcriptomics database’ and cell lines expressing mRNA levels of PPP2R5D greater than 2.5log₂(TPM +1) were deemed suitable for experiments. Does this mean that the only cell line identified with this cut off was HAP1? In my opinion, an important limitation of this study is to have only tested one pair of isogenic models (WT vs KO). Please elaborate. At a minimum, this should be mentioned as a limitation.
Response: From analyzing the public expression database on Depmap, we found that HAP1 expressed PPP2R5D transcript at 5.7 log₂ (TPM+1), which is significantly above the minimum threshold level we’ve established. From the results, it is evident that the selection of HAP1 as cell line for antibody testing was appropriate, as PPP2R5D was easily detected in the lysates when testing the antibodies by western blot. We acknowledge that antibody characterization data generated by following the described protocols does not provide a comprehensive evaluation of the tested antibodies. However, the strength of our approach lies in the simultaneous testing of multiple antibodies from a variety of commercial sources using KO cell lines, aimed at identifying the most suitable antibodies for specific applications. A detailed description of the orthogonal strategy applied to select cell lines for the study is outlined in our antibody characterization platform, available on Nature Protocols (DOI:10.21203/rs.3.pex-2607/v1).

2. Why only consider cancer cell models? Have the authors considered using other types of cells (patient cells or other?) where expression levels of PPP2R5D vary. This would be important to analyze/highlight antibody sensitivity.
3. Researchers in the field working on PPP2R5D-related neurodevelopmental disorder (MRD35/Jordan’s syndrome) often work with patient cells or other models that better recapitulate MRD35 disease characteristics (PPP2R5D pathogenic variants). Have these 6 antibodies been tested on PPP2R5D mutated cell lines (genetic variants, SNP vs. KO)? If so, do these antibodies recognize mutated PPP2R5D and if not, it may also be important to mention the importance of this in the limitation section.
Response to point 2 & 3: We understand that mutations in the epitope coding sequence or elsewhere in the gene coding for the intended target could affect antibody binding, although we think this would be a rare occurrence. However, this is an inherent limitation of any approach that uses cancer cell lines. We have addressed this limitation in the summary paragraph of the results and discussion section.

4. The 6 antibodies - how were they chosen? Are these 6 antibodies the only commercially available?
Response: The antibodies to be tested are donated by YCharOS partners (https://ycharos.com/partners/), who have access to 80% of renewable antibodies covered in commercial catalogs, the other 20% of antibodies from sources that are not partners, why is why we don’t test them. When the partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences.

5. An important aspect of using high-quality and specific antibodies in research is assuring that the antibody selected does not cross-react with other known isoforms. Have these antibodies been tested for cross-reactivity with other PP2A-B56 family isoforms (e.g., PPP2R5C, PPP2R5B, etc.)?
Response: As indicated on their catalog pages, these antibodies are intended to target PPP2R5D, which is why we have tested them, side-by-side, using a PPP2R5D KO cell line. This allows viewers to determine whether the antibodies are detecting PPP2R5D or identify certain variants which they can then use to further validate for cross-reactivity with other PP2A-B56 isoforms. Unfortunately, this degree of validation is outside the scope of our study but we encourage experts in the field to utilize the validation data provided as a starting point to enhance their PPP2R5D related research.

6. I would suggest presenting Table 2 (antibody selection) after Table 1 in the Results and discussion section (before western blot and Immunofluorescence results).
Response: Thank you for bringing up this concern. The newly submitted version of this article will present Table 2 before presenting the results.

7. In the IP experiments (Figure 2) –Could the authors please expand on why we see a multiple band pattern in Figure 2. Earlier in the manuscript, the authors suggest that different MWs can be observed for PPP2R5D depending on lysis buffer used. However, in my understanding, only the IP lysis buffer was used for IP experiments suggesting that PPP2R5D band should be around 75KDa. Is the multiple band pattern only seen when using gradient gels (4-20%)? And if so, it would be important to elaborate which band corresponds to PPP2R5D as some antibodies enrich bottom bands, and others, top bands. Please elaborate on non-specificity or possible cleavage products/degradation as this does not seem to have been tested.
Response: When carrying out the IP experiment (Figure 2), the lysates were freshly prepared and antibodies were immediately processed for IP, which was not the case for the lysis buffer comparison experiment (Figure 1B). In Figure 1, frozen lysates were used to carry out the experiments (A and B). This can explain the difference in signal pattern seen in Figure 2 vs Figure 1B.

8. In the Discussion section, it would be important to clearly summarize which of the 6 antibodies the authors identified as high-quality antibodies that successfully detect PPP2R5D (and which ones are more suitable for protocols tested (WB vs. IP vs. IF)) as this crucial information is missing.
Response: As described in the final paragraphs of the results and discussion section, the authors do not participate in results analysis nor offer explicit antibody recommendations, therefore preventing them from identifying which antibodies were high-performing in each respective application. Due to the confines of the experimental setup, the factors that can influence the performance of antibodies and cell line used, we do not draw conclusions. We leave this analysis up to the viewers and within our gateway we feature an editorial that can be used as a guide on how the scientific community can utilize and analyze the YCharOS data to identify high-performing antibodies within the confines of our protocol (DOI: 10.12688/f1000research.141719.1).
Furthermore, given that that the article is formatted as a Data Note, it does not require the results to be discussed or concluded.

View more View less

Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

[1] 1. Tung HY, Alemany S, Cohen P: The protein phosphatases involved in cellular regulation. 2. Purification, subunit structure and properties of protein phosphatases-2A0, 2A1, and 2A2 from rabbit skeletal muscle. Eur. J. Biochem. 1985; 148(2): 253–263. Publisher Full Text

[2] 2. Alberts AS, Thorburn AM, Shenolikar S, et al.: Regulation of cell cycle progression and nuclear affinity of the retinoblastoma protein by protein phosphatases. Proc. Natl. Acad. Sci. U. S. A. 1993; 90(2): 388–392. PubMed Abstract | Publisher Full Text | Free Full Text

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A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence

Abstract

Keywords

Revised Amendments from Version 1

Introduction

Results and discussion

Table 1. Summary of the cell lines used.

Table 2. Summary of the PPP2R5D antibodies tested.

Figure 1. PPP2R5D antibody screening by Western Blot.

Figure 2. PPP2R5D antibody screening by immunoprecipitation.

Figure 3. PPP2R5D antibody screening by immunofluorescence.

Methods

Antibodies

Cell culture

Antibody screening by Western Blot

Antibody screening by immunoprecipitation

Antibody screening by immunofluorescence

Data availability

Underlying data

Acknowledgment

References

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