F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.133696.2 Data Note Articles Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence

[version 2; peer review: 2 approved]

 Ayoubi Riham Investigation Methodology Visualization 1 Fotouhi Maryam Investigation 1 Southern Kathleen Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4125-3608 1 McPherson Peter S. Conceptualization Funding Acquisition Resources Supervision 1 Laflamme Carl Conceptualization Formal Analysis Funding Acquisition Methodology Project Administration Resources Supervision Validation Writing – Review & Editing https://orcid.org/0000-0002-4125-3608 a 1 NeuroSGC/YCharOS/EDDU collaborative group ABIF consortium Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Québec, H3A 2B4, Canada carl.laflamme@mcgill.ca

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.

 16 10 2023 2023 12 452 9 10 2023 Copyright: © 2023 Ayoubi R et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Vacuolar protein sorting-associated protein 35 is a subunit of the retromer complex, a vital constituent of the endosomal protein sorting pathway. The D620N mutation in the VPS35 gene has been reported to be linked to type 17 Parkinson’s Disease progression, the exact molecular mechanism remains to be solved. The scientific community would benefit from the accessibility of validated and high-quality anti-hVPS35 antibodies. In this study, we characterized thirteen hVPS35 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. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

 Uniprot ID Q96QK1 VPS35 hVPS35 Vacuolar protein sorting-associated protein 35 antibody characterization antibody validation Western Blot immunoprecipitation immunofluorescence Genome Quebec OGI-210 Mitacs Ontario Genomics OGI-210 Michael J. Fox Foundation for Parkinson’s Research 18331 Genome Canada OGI-210 This work was supported by the Michael J. Fox Foundation for Parkinson’s Research (MJFF) (grant no. 18331). The Government of Canada through Genome Canada, Genome Quebec and Ontario Genomics (grant no. OGI-210). The Structural Genomics Consortium is a registered charity (no. 1097737) that receives funds 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. RA is supported by Mitacs fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

In the introduction section of this revised article, we have incorporated additional comprehensive information about PARK17, along with an explanation of what a high-performing antibody is in each tested application. To the results and discussion section, we have included a description as to why the authors and YCharOS initiative does not score nor interpret antibody characterization data.

 Introduction

Vacuolar protein sorting-associated protein 35 (hVPS35) is a component of the retromer complex. 1 The retromer complex is a multimeric protein complex responsible for sorting transmembrane cargo from the endosome to the trans-Golgi network or plasma membrane, a pathway which is conserved across all eukaryotes. 1 , 2 Composed of two major subcomplexes, the cargo-selective complex and the membrane bound vacuolar protein sorting-associated protein (VPS) complex, hVPS35 serves as an essential component of the VPS complex where it mediates the recruitment of the retromer complex to the endosomal membrane. 1

There are 23 PARK genes which have been discovered through genome-wide association studies, 23 Parkinson’s disease (PD)-associated PARK genes have been discovered, namely PRKN, PINK2, LRRK2, SNCA, VPS35 and others. 3 – 6 Variants of the VPS35 gene, which is PARK17, have recently been associated with the development of familial PD, among other neurodegenerative diseases. 7 – 9 A missense mutation in the gene, D620N, has been reported in numerous individuals and families with PD. 10 – 16 Further research is required to investigate the molecular mechanisms in which VPS35 mutations induces neurodegeneration in PD. 7

Mechanistic studies would be greatly facilitated with the availability of high-performing antibodies. Under our standardized procedure, a high-performing antibody, or a successful antibody, can be defined according to its application. In Western blot, a high-performing antibody will specifically immunodetects the target protein in the Wild-type (WT) but not in the knockout (KO) lysate. In immunoprecipitation, a high-performing antibody immunocaptures the target protein to at least 10% of the starting material. For immunofluorescence, a high-performing antibody immunolocalizes the target protein by generating a fluorescent signal that is 1.5-fold higher in WT cells than in the KO cells. 17

Here, we compared the performance of a range of commercially-available antibodies for hVPS35 and identified high-performing antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of hVPS35 properties and function.

 Results and discussion

Our standard protocol involves comparing readouts from wild-type (WT) and knockout (KO) cells. 17 – 23 The first step was to identify a cell line(s) that expresses sufficient levels of hVPS35 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 log 2 (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 hVPS35 transcript at RNA levels above the average range of cancer cells analyzed. Parental and VPS35 KO HAP1 cells were obtained from Horizon Discovery ( 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 HZGHC000863c012 CVCL_TX57 HAP1 VPS35 KO

For Western Blot experiments, we resolved proteins from WT and VPS35 KO cell extracts and probed them side-by-side with all antibodies in parallel ( Figure 1). 19 – 23

hVPS35 antibody screening by Western Blot.

Lysates of HAP1 (WT and VPS35 KO) were prepared and 20 μg of protein were processed for Western Blot with the indicated hVPS35 antibodies. The Ponceau stained transfers of each blot are presented to show equal loading of WT and KO lysates and protein transfer efficiency from the polyacrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. An exception was given for antibody 81453**, which was titrated to 1/500, as the signal was too weak when following the supplier’s recommendations. Antibody dilution used: GTX635821** at 1/1000, GTX108058 at 1/1000, GTX116260 at 1/1000, A9278** at 1/1000, MA5-34647** at 1/1000, PA5-21898 at 1/1000, PA5-30654 at 1/1000, ab157220** at 1/1000, ab57632* at 1/270, ab118838 at 1/900, 81453** at 1/500, 10236-1-AP at 1/500, NBP2-75710** at 1/1000. Predicted band size: 91 kDa. *=monoclonal antibody, **=recombinant antibody.

For immunoprecipitation experiments, we used the antibodies to immunopurify hVPS35 from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the hVPS35 protein in extracts, in the immunodepleted extracts and in the immunoprecipitates ( Figure 2). 19 – 23

hVPS35 antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated hVPS35 antibodies pre-coupled to Dynabeads protein A or protein G. Samples were washed and processed for Western Blot with the indicated hVPS35 antibody. For Western Blot, GTX635821** and 81453** were used at 1/1000 and 1/500, respectively. The Ponceau stained transfers of each blot are shown. SM=2% starting material; UB=2% unbound fraction; IP=immunoprecipitate. *=monoclonal antibody, **=recombinant antibody.

For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 24 In brief, we plated WT and KO cells together in the same well and imaged both cell types in the same field of view to reduce staining, imaging and image analysis bias ( Figure 3).

hVPS35 antibody screening by immunofluorescence.

HAP1 WT and VPS35 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 a glass bottom. Cells were stained with the indicated hVPS35 antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (WT), red (antibody staining) and far-red (KO) channels was performed. Representative images of the merged blue and red (grayscale) channels are shown. WT and KO cells are outlined with green and magenta dashed line, respectively. Antibody dilutions were chosen according to the recommendations of the antibody supplier. When the concentration was not indicated by the supplier, which was the case for antibodies GTX116260, A9278**, MA5-34647**, PA5-30654, 81453** and NBP2-75710**, we tested the antibodies at 1/600, 1/400, 1/1000, 1/60, 1/60 and 1/500, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: GTX635821** at 1/1000, GTX108058 at 1/600, GTX116260 at 1/600, A9278** at 1/400, MA5-34647** at 1/1000, PA5-21898 at 1/100, PA5-30654 at 1/60, ab157220** at 1/500, ab57632* at 1/1000, ab118838 at 1/1000, 81453** at 1/60, 10236-1-AP at 1/50, NBP2-75710** at 1/500. Bars = 10 μm. *=monoclonal antibody, **=recombinant antibody.

In conclusion, we have screened hVPS35 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions. Under our standardized experimental conditions, several high-quality antibodies were identified, 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. 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.

The underlying data can be found of Zenodo. 25 , 26

Methods Antibodies

All hVPS35 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 27 Peroxidase-conjugated goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number 62-6120 and 65-6520). Alexa-555-conjugated goat anti-rabbit and anti-mouse secondary antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424).

Summary of the hVPS35 antibodies tested.
Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μL) Vendors recommended applications
GeneTex GTX635821 ** 44151 AB_2888583 recombinant-mono HL1017 rabbit 1.00 Wb, IF
GeneTex GTX108058 43950 AB_1241448 polyclonal - rabbit 0.66 Wb, IF
GeneTex GTX116260 40282 AB_10626418 polyclonal - rabbit 0.64 Wb, IP
Abclonal A9278 ** 4000001509 AB_2863704 recombinant-mono ARC1509 rabbit 0.37 Wb
Thermo Fisher Scientific MA5-34647 ** XD3571015 AB_2848555 recombinant-mono JB33-82 rabbit 1.00 Wb
Thermo Fisher Scientific PA5-21898 XE3572719 AB_11153540 polyclonal - rabbit 0.66 Wb, IF
Thermo Fisher Scientific PA5-30654 XD3572719D AB_2548128 polyclonal - rabbit 0.64 Wb, IP
Abcam ab157220 ** GR117932 AB_2636885 recombinant-mono EPR11501(B) rabbit 0.11 Wb, IF
Abcam ab57632 * GR3330429 AB_946126 monoclonal 2D3 mouse 0.27 Wb
Abcam ab118838 GR250228 AB_2923524 1 polyclonal - rabbit 0.90 Wb, IF
Cell Signaling Technology 81453 ** 1 AB_2923525 1 recombinant-mono E6S4l rabbit 0.06 Wb, IP
Proteintech 10236-1-AP 22564 AB_2215216 polyclonal - rabbit 0.26 Wb, IP, IF
Bio-Techne NBP2-75710 ** HP0606 AB_2923523 1 recombinant-mono JB33-82 rabbit 1.00 Wb

Wb=Western blot; IF=immunofluorescence; IP=immunoprecipitation.

=monoclonal antibody.

=recombinant antibody.

refers to RRID recently added to the Antibody Registry (on February 2023), they will be available on their website in the coming weeks.

 Cell culture

Both HAP1 WT and VPS35 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 28 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

Western Blots were performed as described in our standard operating procedure. 29 HAP1 WT and VPS35 KO were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. number 89901) supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat. number 78429). Lysates were sonicated briefly and incubated for 30 min on ice. Lysates were spun at ~110,000 x 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 pre-cast mini 4-15% gradient polyacrylamide gels from Bio-Rad (cat. number 4561084) and transferred onto 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% bovine serum albumin (BSA) (Wisent, cat. number 800-095) in TBS with 0,1% Tween 20 (TBST) (Cell Signaling 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 incubated with Pierce ECL from Thermo Fisher Scientific (cat. number 32106) prior to detection with the HyBlot CL autoradiography films from Denville (cat. number 1159T41).

 Antibody screening by immunoprecipitation

Immunoprecipitation was performed as described in our standard operating procedure. 30 Antibody-bead conjugates were prepared by adding 2 μg to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat. number 87788) in a 1.5 mL microcentrifuge tube, together with 30μL of Dynabeads protein A- (for rabbit antibodies) or protein G- (for mouse antibodies) 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) (Thermo Fisher Scientific, cat. number 87788) supplemented with protease inhibitor (MilliporeSigma, cat. number P8340). Lysates were rocked for 30 min at 4°C and spun at 110,000 x 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 ~2 hrs 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 pre-cast mini 4-15% gradient polyacrylamide gels. Prot-A: HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a concentration of 0.3 μg/mL.

 Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure. 19 – 24 HAP1 WT and VPS35 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 plated in 96 well glass plates (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 oC, 5% CO 2. Cells were fixed in paraformaldehyde (PFA) (Beantown chemical, cat. number 140770-10ml) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 15 min at room temperature and then washed 3 times with PBS. 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% BSA, 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 hVPS35 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 20x/0.95 NA water objective lens and scientific CMOS camera (16-bit, 1.97mm 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 x 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 hVPS35, https://doi.org/10.5281/zenodo.7671730. 25

Zenodo: Dataset for the hVPS35 antibody screening study, https://doi.org/10.5281/zenodo.7795779. 26

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: Riham Ayoubi, Thomas M. Durcan, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch and Kathleen Southern.

ABIF consortium: Claire M. Brown and Joel Ryan.

Thank you to the Michael J. Fox Foundation for Parkinson’s Research for their contribution to the project, the Characterization of Antibody Reagents for 15 Diverse Protein Targets.

An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.7671730)

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Methods. 2021;18(1):100–106. 33318659 10.1038/s41592-020-01018-x 10.5256/f1000research.157173.r234849 Reviewer response for version 2 Sontag Emily 1 Referee https://orcid.org/0000-0003-3068-6253 Marquette University, Milwaukee, Wisconsin, USA

Competing interests: No competing interests were disclosed.

 9 2 2024 Copyright: © 2024 Sontag E 2024 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

The article characterizes 13 different commercial antibodies to the human VPS35 protein, which is associated with Parkinson's disease. This information is highly valuable to the field, but a few clarifications will assist readers in interpreting the data and using it to make decisions on which reagents to use in their own experiments. 

The amendments provide additional information about PARK17, but introduce a bit of confusion. In the third sentence of the first paragraph of the introduction, it is unclear if the retromer complex or the hVPS35 is composed of 2 subcomplexes. Additionally, the first sentence of the second paragraph of the introduction is confusing as it appears that the sentence is repeated within itself. The definition of a high-performance for each application is very clearly stated. 

While the authors are clear on why they do not offer analyses or recommendations, it would be very helpful to include quantification of the Western blots from the IP experiments and the intensity values of the immunofluorescence. Several of the antibodies do not appear to meet the threshold for high-performance by those techniques (10% of input and a 1.5-fold increase in signal, respectively). The measured values would aid experts in interpreting the data and determining which antibodies meet the standard of a high-performing antibody.

Additionally, it is unclear why the GTX635821 and 81453 antibodies were chosen for the Western blot analysis of the IP experiments. The NBP2-75710 antibody appears to give the best signal on the Western blot screening in Figure 1. The legend for Figure 3 states that the representative images of the blue and red channels are merged, but they are shown as separate grayscale images. 

The article is scientifically sound and contributes to the field, but could be improved with minor revisions.

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

Yes

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

Yes

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

Partly

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Biochemistry, Cell Biology, protein homeostasis, ESCRT and VPS protein function.

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.

 10.5256/f1000research.157173.r215700 Reviewer response for version 2 Marzolo Maria-Paz 1 2 Referee https://orcid.org/0000-0002-2328-2464 Pontificia Universidad Catolica de Chile, Santiago, Santiago Metropolitan Region, Chile Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Catolica de Chile, Santiago, Santiago Metropolitan Region, Chile

Competing interests: No competing interests were disclosed.

 21 12 2023 Copyright: © 2023 Marzolo MP 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve

In this new revised version, the authors have included the references suggested, and they have improved the introduction with a detailed explanation of retromer complex, its implication in Parkinson´s disease, and the definition of a high-performing antibody. The section is now clear and complete.

This is a well-performed study, with relevant data to be considered for researchers working on retromer functions and expression, which, as was suggested by our initial review, would benefit by including more than one cell line. However, the authors explain the study's limitations in terms of using only one cell line. We think the study has value in itself and contributes to the field.

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

Partly

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

Partly

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

Partly

Are the protocols appropriate and is the work technically sound?

Partly

Reviewer Expertise:

Cell Biology, neurobiology, membrane trafficking, cell polarity, signaling pathways.

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.

 10.5256/f1000research.146706.r191098 Reviewer response for version 1 Marzolo Maria-Paz 1 2 Referee https://orcid.org/0000-0002-2328-2464 Macias Vania 3 Co-referee Pontificia Universidad Catolica de Chile, Santiago, Santiago Metropolitan Region, Chile Laboratorio de Tráfico Intracelular y Señalización, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Catolica de Chile, Santiago, Santiago Metropolitan Region, Chile Cell and Molecular Biology, Pontifical Catholic University of Chile, Santiago, Santiago Metropolitan Region, Chile

Competing interests: No competing interests were disclosed.

 7 9 2023 Copyright: © 2023 Marzolo MP and Macias V 2023 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

The article is a data note that evaluates different commercial antibodies for the human Vacuolar protein sorting-associated protein 35 (hVPS35),a. subunit of the Retromer complex, also composed of VPS26 and VPS29 subunits. Retromer is involved in several cargo sorting processes at the endosomes, regulating endosome to plasma membrane trafficking of cargoes and the endosome to Golgi apparatus retrograde transport of cargoes such as the mannose-6-phosphate receptors. Mutations in Retromer are associated with neurodegenerative conditions, such as Parkinson's Disease (PD). This article is particularly relevant as a data note for evaluating different commercial antibodies for VPS35 detection, but some mistakes should be addressed.

In the Abstract and the Introduction, it is mentioned a type 17 Parkinson's disease progression. The VPS35 gene is the PARK17, but there are not 17 types of PD. The authors could check the PARK genes that historically are the genes that have been linked to PD. Reference 1 can be complemented with other articles; I suggest including the articles:

Haft, C. R., de la Luz Sierra, M., Bafford, R., Lesniak, M. A., Barr, V. A., & Taylor, S. I. (2000). Human orthologs of yeast vacuolar protein sorting proteins Vps 26, 29, and 35: Assembly into multimeric complexes. Molecular Biology of the Cell, 11, 4105–4116.

Seaman, M. N. J., Marcusson, E. G., Cereghino, J. L., & Emr, S. D. (1997). Endosome to Golgi retrieval of the vacuolar protein sorting receptor,

Vps10p requires the function of the VPS29, VPS30, and VPS35 gene products. Journal of Cell Biology, 137, 79–92.

Seaman, M. N. J., McCaffery, J. M., & Emr, S. D. (1998). A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. Journal of Cell Biology, 142, 665–681.

Seaman, M. N. J. (2004). Cargo-selective endosomal sorting for retrieval to the Golgi requires a retromer. Journal of Cell Biology, 165, 111–122.  https://doi.org/10.1083/jcb.20031 2034

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

The study is relevant to the field. It is necessary to characterize and find reliable antibodies that can detect the VPS35, without false positives, for describing and ascribing different and eventually new retromer functions and cargoes. The authors established that the main AIM of the data note is to use it as a guide to select the appropriate antibody for the research. The guide could help scientists in the field to invest in quality antibodies, saving time, money, and other resources in the characterization of antibodies that, in the end, will be useless. This AIM is accomplished; as a guide, this article gives useful information. However, it could be improved by discussing the reliability of the antibodies tested and the pertinence of the results obtained.

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

The authors analyze the antibody's performance in three techniques widely utilized in cell biology: western blot, immunoprecipitation, and immunofluorescence. These are techniques often used in the field, including membrane trafficking studies and neurobiology studies. Nevertheless, the results could be improved.

The authors decided to test the antibodies in the carcinogenic cell line HAP1 should explore other non-carcinogenic cell lines as well as neuronal cell lines considering that many mutants of VPS35 are associated with neurodegenerative diseases, including PD. Moreover, even among cells of the same species, antibody performance also depends on the cellular type; therefore, it would be desirable to test the antibodies in a neuron-like cell line as H4, SH-Sy5Y, to mention some.

In the western blot experiments, there are unspecific bands in the WT and KO lines for GTX116260, PA5-30654, and ab57632*. This should be remarked in an additional table or Table 2 for scientists to evaluate the pertinence of these antibodies.

In the immunofluorescence, the signal shown does not correlate with an expected signal of Retromer following the VPS35 subunit. It should show a vesicular pattern with identifiable structures. This could be correlated with other endosome markers or cargos such as mannose-6-phosphate, the GTPase Rab7, or the expression of a GFP-tag VPS35. Besides, authors should consider testing other fixations as methanol and performing negative controls in non-permeabilized cells. I wonder if 20X is the ideal magnification for the analysis because the label is very diffused. However, comparing the WT and KO lines in the same field is a great idea.

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

The methods are clear and detailed. However, the authors could explain why, for the western blots, they decided to block with milk and then incubate the primary antibody in BSA. For example. for some antibodies, the BSA is also required for blocking. A clarification on the concept of high-performing antibodies and what it entails should be included.

Are the datasets presented in a usable and accessible format?

The datasets are clearly and accessibly presented, but Table 2 could include the performance of the antibodies in the different techniques and if the antibodies have been cited in other articles.

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

Partly

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

Partly

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

Partly

Are the protocols appropriate and is the work technically sound?

Partly

Reviewer Expertise:

Cell Biology, neurobiology, membrane trafficking, cell polarity, signaling pathways.

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, however we have significant reservations, as outlined above.

References : Human orthologs of yeast vacuolar protein sorting proteins Vps26, 29, and 35: assembly into multimeric complexes. Mol Biol Cell .2000;11(12) : 10.1091/mbc.11.12.4105 4105-16 11102511 10.1091/mbc.11.12.4105 : Endosome to Golgi retrieval of the vacuolar protein sorting receptor,Vps10p requires the function of the VPS29, VPS30, and VPS35 gene products. Journal of Cell Biology .1997;79-92 : A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J Cell Biol .1998;142(3) : 10.1083/jcb.142.3.665 665-81 9700157 10.1083/jcb.142.3.665 : Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer. J Cell Biol .2004;165(1) : 10.1083/jcb.200312034 111-22 15078902 10.1083/jcb.200312034 Southern Kathleen McGill University, Canada

Competing interests: No competing interests were disclosed.

 6 10 2023

Thank you for your review and feedback. We will be addressing your comments and submitting a new version of the article, with many of your suggestions included.

To the abstract and introduction, we have removed the mention of type 17 Parkinson’s disease. Furthermore, in the introduction we have included information to mention other PARK genes that have been identified in research, including PARK17. We are always looking to improve on our articles and hope this satisfies your feedback!

In regard to your comment on the rationale for creating the dataset, we understand that you believe it would enhance the quality of the article if we discussed the performance of the antibodies tested and reported their specificity or selectivity in regards to the results. In response to this feedback, it’s important to clarify that YCharOs does not engage in result analysis nor do we offer explicit antibody recommendations. We’re not experts of these proteins and prefer to remain impartial which is why we carry out the study without interpreting the results. The purpose of YCharOS is to deliver antibody characterization reports as a public good collection to benefit biomedical research as a whole. It is for this reason that we chose to publish our articles using the Data Note format, as it does not require result analysis but rather presents KO characterization data for experts of the individual targets to further their studies and projects. We have found that, for the most part, scientists viewing our articles have the expertise to the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs. We understand that this point has not been made clear within the article, which is why we have added a new paragraph to the Results and Discussion section, explaining our reasonings as to why we do not recommend or score the antibodies tested.

In terms of cell lines, at this point, testing and validating the antibodies in various neuronal cell lines is outside the scope of our standard protocol and study. We utilize the Cancer Dependency Map (DepMap) as a guide to select a cell line with adequate levels of protein of interest. That being said, we hope to revisit all Parkinson’s disease- related protein targets in the future by quantifying the antibody performance on a larger scale and analyzing protein expression in various neuronal cell lines.

In terms of evaluating the antibodies you mentioned in Western blot, as previously mentioned, we believe scientists viewing the article will have the expertise to see that the antibodies mentioned are non-selective using our current experimental set-up.

The reason as to why different fixation methods were not used, is because with our current workflow we identified antibodies that specifically target hVPS35 by immunofluorescence (IF). It is only in the case that no successful antibodies were identified that we consider other fixation protocols such as methanol.

Furthermore, our goal for the IF application is not to identify the subcellular distribution of hVPS35, but rather demonstrate to the scientific community which of the antibodies perform successfully in IF. Meaning, we look for antibodies that immunolocalizes the target protein by generating a fluorescence signal in WT cells that is at least 1.5-fold higher than the signal in KO cells (1). Using a 20X magnification allows us to evaluate the antibodies selectivity but does not have the resolution to dive into the subcellular distribution of the antibodies. With our reports, the experts now know which antibodies are successful in identifying VPS35, and can further their research by identifying subcellular distribution utilizing organelle markers.

We intend on clarifying this factor in our FAQ section on our YCharOS gateway.

After performing our standard operating procedure for around 80 protein targets and 800 antibodies, we haven’t seen a difference in results when blocking with milk or BSA. Furthermore, Milk was chosen as a blocking solution as it doesn’t get contaminated as easily, allowing us to repeat the experiment, if necessary. 

We agree that the definition of a high-performing antibody is not made evident in the first version of the article. In the introduction of the new article version to be submitted shortly, we have included a small description of what high-performing antibody signifies in each application. You can also visit an article written by Riham et al. which provides an in-depth description on what a successful antibody looks like in each application (Box 1) (1)

We don’t look at antibody performance in individual reports, but we do in a separate study, looking at the overall performance of antibodies, compared to their use in literature (1).

1. Ayoubi R, Ryan J, Biddle MS, Alshafie W, Fotouhi M, Bolivar SG, et al. Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. eLife. 2023;12:RP91645.