F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.130126.1 Data Note Articles The identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence

[version 1; peer review: 2 approved with reservations]

 Alshafie Walaa Investigation Methodology 1 Ayoubi Riham Investigation Methodology Visualization Writing – Review & Editing 1 Fotouhi Maryam Investigation 1 Southern Kathleen Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0000-0002-4125-3608 1 Laflamme Carl Conceptualization Data Curation Funding Acquisition Methodology Project Administration Resources Supervision Validation Visualization Writing – Review & Editing https://orcid.org/0000-0002-4125-3608 a 1 NeuroSGC/YCharOS collaborative group Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Quebec, 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- Abclonal -Aviva Systems Biology -Bio Techne -Cell Signalling Technology -Developmental Studies Hybridoma Bank -Genetex – Horizon Discovery – Proteintech – Synaptic Systems -Thermo Fisher Scientific.

 13 2 2023 2023 12 172 7 2 2023 Copyright: © 2023 Alshafie W 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.

Moesin is a cytoskeletal adaptor protein, involved in the modification of the actin cytoskeleton, with relevance to Alzheimer’s Disease. Well characterized anti-Moesin antibodies would benefit the scientific community. In this study, we characterized ten commercial antibodies for Moesin in 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 P26038 MSN Moesin antibody characterization antibody validation Western Blot immunoprecipitation immunofluorescence Ontario Genomics OGI-210 Mitacs postdoctoralfellowship National Institutes of Health RF1AG057443 Genome Canada OGI-210 National Institute of Aging U54AG065187 This work was supported by a grant from the National Institute of Health (NIH) to improve the reliability of AMP-AD target hypotheses (no. RF1AG057443) and the Emory-Sage-SGC TREAT-AD center established by the National Institutes of Aging (no. U54AG065187). The Government of Canada through Genome Canada and Ontario Genomics (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. WA and RA are supported by a Mitacs postdoctoral fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Moesin is a cytoskeletal adaptor protein that belongs to the Ezrin-Radixin-Moesin family of proteins that connect the actin cytoskeleton to the plasma membrane, regulating the structure and function of specific domains of the cell cortex. 1 , 2 Moesin plays a pertinent role in immunity, acting on T and B-cell homeostasis and self-tolerance. 3 , 4

Proteomic and protein co-expression network analysis of Alzheimer's Disease (AD) brain has revealed a module that is enriched in inflammation-related proteins. 5 Moesin, along with CD44 antigen, have emerged as key drivers in this inflammation module. Disrupting the Moesin-CD44 pathway is a current focus in AD research. 6 Mechanistic studies would be greatly facilitated with the availability of high-quality antibodies.

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

 Results and discussion

Our standard protocol involves comparing readouts from wild-type and knockout cells. 7 , 8 The first step is to identify a cell line(s) that expresses sufficient 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 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 HeLa cells expressed the Moesin transcript at RNA levels above the average range of cancer cells analyzed. Parental and MSN knockout HeLa cells were obtained from Abcam ( Table 1).

Summary of the cell lines used.
Institution Catalog number RRID (Cellosaurus) Cell line genotype
Abcam ab255448 CVCL_0030 HeLa WT
Abcam ab265020 CVCL_B9VN HeLa MSN KO

For Western Blot, we resolved proteins from wild-type and MSN KO cell extracts and probed them side-by-side with all antibodies in parallel ( Figure 1).

Moesin antibody screening by Western Blot.

Lysates of HeLa (WT and MSN KO) were prepared, and 25 μg of protein were processed for Western Blot with the indicated Moesin 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 acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen based on the recommendations provided by suppliers with exceptions for antibodies MA5-32231**, MA5-17130* and GTX101708, which were titrated as the signal received was too strong following the supplier’s recommendations. The antibody dilutions were as follows: MA5-32231** at 1/5000, MA5-17130* at 1/5000, NBP2-32876* at 1/5000, NBP2-44579* at 1/5000, NBP2-44580* at 1/5000, GTX101708 at 1/5000, ab52490** at 1/1000, ab151542** at 1/1000, ab169789** at 1/10000 and ab193380** at 1/400. Predicted band size: 68 kDa. *Monoclonal antibody, **Recombinant antibody.

For immunoprecipitation, we used the antibodies to immunopurify Moesin from HeLa cell extracts. The performance of each antibody was evaluated by detecting the Moesin protein in extracts, in the immunodepleted extracts and in the immunoprecipitates ( Figure 2).

Moesin antibody screening by immunoprecipitation.

HeLa lysates were prepared, and IP was performed using 1.0 μg of the indicated Moesin antibodies pre-coupled to either protein G or protein A Sepharose beads. Samples were washed and processed for Western Blot with the indicated Moesin antibody. For immunoblot, NBP2-44579*, GTX101708 and ab169789** were used at 1/20000, 1/20000 and 1/10000, respectively. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM = 10% starting material; UB = 10% unbound fraction; IP = immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 9 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 imaging and analysis bias ( Figure 3).

Moesin antibody screening by immunofluorescence.

HeLa WT and MSN 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 on coverslips. Cells were stained with the indicated Moesin 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 yellow and magenta dashed line, respectively. Antibody dilutions were chosen based on supplier recommendations, except for MA5-32231** which was titrated to 1/1000 as the signal was too strong. When concentrations were not provided by the supplier, antibodies were tested at 1/1000, which was the case for MA5-17130*. Antibody dilutions used; MA5-32231** at 1/1000; MA5-17130* at 1/1000; NBP2-32876* at 1/200; NBP2-44579* at 1/200; NBP2-44580* at 1/200; GTX101708 at 1/200; ab52490** at 1/200, ab151542** at 1/200, ab169789** at 1/100, ab193380** at 1/200. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

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

 Methods Antibodies

All Moesin antibodies are listed in Table 2. 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).

Summary of Moesin antibodies tested.
Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications
Thermo Fisher Scientific MA5-32231** VJ3101165 AB_2809517 recombinant-mono SC69-01 rabbit 1.00 Wb, IF
Thermo Fisher Scientific MA5-17130* VJ3101185 AB_2538601 monoclonal 2C12 mouse 1.00 Wb
Bio-Techne NBP2-32876* 4478-1XP160531 AB_2885048 monoclonal SPM562 mouse 0.20 Wb, IF
Bio-Techne NBP2-44579* 44578-2P190315 AB_2885047 monoclonal MSN/492 mouse 0.20 Wb, IF
Bio-Techne NBP2-44580* 4478-3P190605 AB_2885046 monoclonal MSN/493 mouse 0.20 Wb, IF
GeneTex GTX101708 40198 AB_10618789 polyclonal - rabbit 0.21 Wb, IP, IF
Abcam ab52490** GR3207377-11 AB_881245 recombinant-mono EP1863Y rabbit 0.20 Wb, IP, IF
Abcam ab151542** GR112662-8 AB_2893185 recombinant-mono EPR2428(2) rabbit 0.09 Wb, IF
Abcam ab169789** GR121830-3 AB_2885098 recombinant-mono EPR2429(2) rabbit 0.07 Wb, IP, IF
Abcam ab193380* GR3373113-1 AB_2885109 monoclonal MSN/491 mouse 0.20 Wb, IP

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

 Cell culture

HeLa WT and MSN KO cells used are listed in Table 1. Cells were cultured in DMEM high-glucose (GE Healthcare cat. number SH30081.01) containing 10% fetal bovine serum (GE Healthcare cat. Number SH30072.03), 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. 10 HeLa WT and MSN KO cells were collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1.0 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitor. Lysates were sonicated briefly and incubated 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.

Western Blots were performed with large 4–15% gradient polyacrylamide gels and transferred on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western Blot. Blots were blocked with 5% milk for 1 hr, and antibodies were incubated O/N at 4°C with 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST). 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 are incubated with ECL from Pierce (cat. number 32106) prior to detection with HyBlot CL autoradiography films from Denville (cat. number 1159T41).

 Antibody screening by immunoprecipitation

Immunoprecipitation was performed as described in our standard operating procedure. 11 Antibody-bead conjugates were prepared by adding 1.0 μg of antibody to 500 μl of PBS with 0.01% triton X-100 in a microcentrifuge tube, together with 30 μl of protein A - (for rabbit antibodies) or protein G - (for mouse antibodies) Sepharose beads. Tubes were rocked overnight at 4°C followed by two washes to remove unbound antibodies.

HeLa cells were collected in HEPES buffer (20 mM HEPES, 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, pH 7.4) supplemented with protease inhibitor. Lysates are rocked 30 min at 4°C and spun at 110,000 × g for 15 min at 4°C. One ml aliquots at 1 mg/ml of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C. Following centrifugation, the unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of HEPES lysis buffer and processed for SDS-PAGE and Western Blot on a 4-15% acrylamide gel.

 Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure. 9 HeLa cells (WT and MSN KO) were labelled with a green dye and with a deep red fluorescent dye from Abcam (cat. number ab176735 and ab176736), respectively. WT and KO cells were plated on glass coverslips as a mosaic and incubated for 24 hrs in a cell culture incubator. Cells were fixed in 4% PFA (in PBS) for 15 min at room temperature and then washed 3 times with PBS. Cells were permeabilized in PBS with 0.1% Triton X-100 for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum and 0.01% Triton X-100 for 30 min at room temperature. Coverslips were incubated face down on a 50 μl drop (on paraffin film in a moist chamber) with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary Moesin antibodies O/N at 4°C. Cells were washed 3 times for 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies, including DAPI, in IF buffer at a dilution of 1.0 μg/ml for 1 hr at room temperature. Cells were washed 3 times for 10 min with IF buffer and once with PBS. Coverslips were mounted on a microscopic slide using fluorescence mounting media (DAKO).

Imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apo 40× oil objective (NA = 1.40). Analysis was done using Image J. All cell images represent a single focal plane. Figures were prepared using Adobe Photoshop to adjust contrast, apply 1 pixel Gaussian blur and then assembled with Adobe Illustrator.

 Data availability Underlying data

Zenodo: Antibody Characterization Report for Moesin, DOI: https://doi.org/10.5281/zenodo.4724169. 12

Zenodo: Dataset for the Moesin antibody screening study, DOI: https://doi.org/10.5281/zenodo.7566164. 13

This project contains the following data:

Head to head comparison of available commercial antibodies against Moesin by immunoblot (Western blot), immunoprecipitation and immunofluorescence.

This project contains the following underlying data included in a study aiming at characterizing antibodies for the Moesin protein. The study is available on Zenodo ( https://doi.org/10.5281/zenodo.4724169). 12

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

 Acknowledgment

We’d like to thank the NeuroSGC/YCharOS collaborative group for their important contributions to the creation of an open scientific ecosystem of antibody manufacturers and knockout cell line suppliers as well as the development of community-agreed protocols. Members of the group can be found below.

NeuroSGC/YCharOS collaborative group: Riham Ayoubi, Aled M. Edwards, Carl Laflamme, Peter S. McPherson, Chetan Raina and Kathleen Southern.

An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.4724169). 12

References Huang L Wong TY Lin RC : Replacement of threonine 558, a critical site of phosphorylation of moesin in vivo, with aspartate activates F-actin binding of moesin. Regulation by conformational change. J. Biol. Chem. 1999;274(18):12803–12810. 10212266 10.1074/jbc.274.18.12803 Tsukita S Yonemura S : ERM (ezrin/radixin/moesin) family: from cytoskeleton to signal transduction. Curr. Opin. Cell Biol. 1997;9(1):70–75. 10.1016/S0955-0674(97)80154-8 Serrador JM Alonso-Lebrero JL Pozo MA del : Moesin interacts with the cytoplasmic region of intercellular adhesion molecule-3 and is redistributed to the uropod of T lymphocytes during cell polarization. J. Cell Biol. 1997;138(6):1409–1423. 9298994 10.1083/jcb.138.6.1409 PMC2132557 Serrador JM Nieto M Alonso-Lebrero JL : CD43 interacts with moesin and ezrin and regulates its redistribution to the uropods of T lymphocytes at the cell-cell contacts. Blood. 1998;91(12):4632–4644. 9616160 10.1182/blood.V91.12.4632 Seyfried NT Dammer EB Swarup V : A Multi-network Approach Identifies Protein-Specific Co-expression in Asymptomatic and Symptomatic Alzheimer's Disease. Cell Syst. 2017;4(1):60–72.e4. 27989508 10.1016/j.cels.2016.11.006 PMC5269514 Katis V Bradshaw W Betarbet R : Targeting the Moesin-CD44 pathway for therapeutic intervention in Alzheimer’s disease: Molecular and cell biology/protein-protein interactions. Alzheimers Dement. 2020;16:e045915. 10.1002/alz.045915 Laflamme C McKeever PM Kumar R : Implementation of an antibody characterization procedure and application to the major ALS/FTD disease gene C9ORF72. elife. 2019;8:8. 10.7554/eLife.48363 Alshafie W Fotouhi M Shlaifer I : Identification of highly specific antibodies for Serine/threonine-protein kinase TBK1 for use in immunoblot, immunoprecipitation and immunofluorescence. F1000Res. 2022;11:977. 10.12688/f1000research.124632.1 Alshafie W McPherson P Laflamme C : Antibody screening by Immunofluorescence. 2021. Ayoubi R McPherson PS Laflamme C : Antibody Screening by Immunoblot. 2021. Ayoubi R Fotouhi M McPherson P : Antibody screening by Immunoprecitation. 2021. Alshafie W Ayoubi R Fotouhi M : Antibody Characterization Report for Moesin. 2021. 10.5281/zenodo.4724169 Laflamme C : Dataset for the Moesin antibody screening study.[Data set]. Zenodo. 2023. 10.5281/zenodo.7566164 10.5256/f1000research.142858.r177143 Reviewer response for version 1 Na Sungsoo 1 Referee Indiana University Purdue University Indianapolis, Indianapolis, Indiana, USA

Competing interests: No competing interests were disclosed.

 2 8 2023 Copyright: © 2023 Na S 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

This manuscript compared the commercially available antibodies through various assays, including Western blot, immunoprecipation, and immunofluorescence. All the experimental methods and data are well presented. However, this manuscript lacks descriptions on the comparison, characterization, and identification of the antibodies in the results section although the authors claimed that this manuscript is a "guide to select the most appropriate antibody for their specific needs".

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

Partly

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:

live cell imaging, cell signaling analysis

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.

Southern Kathleen McGill University, Canada

Competing interests: No competing interests were disclosed.

 3 8 2023

Thank you Sungsoo Na for reviewing this article.

As a third-party entity, YCharOS refers to remain unbiased which is why the authors do not compare the performance of antibodies based on the displayed results. After presenting the YCharOS initiative on several occasions we have found that, for the most part, scientists interested in our reports have the expertise to interpret the results and decipher which antibodies are appropriate for their research needs. Furthermore, these antibodies are tested under one specific condition and scoring the antibodies would only be valid under this setup and cell line used. We believe that the results from Western Blot, immunoprecipitation and immunofluorescence are telling and will help viewers in finding high-quality antibodies for Moesin. For those that have trouble interpreting the results, we have a clear description on what a “successful” antibody looks like in each application on our FAQ page of the YCharOS gateway ( https://f1000research.com/gateways/ycharos/faqs ).

We hope you understand our reasoning for our wish to remain unbiased. If there are any other changes you believe would be appropriate to enhance this manuscript that do not involve comparing or scoring the antibodies based on performance, please let us know and we would be happy to do so.

Thank you again for your feedback.

 10.5256/f1000research.142858.r177135 Reviewer response for version 1 Avery Lyndsay 1 Referee https://orcid.org/0000-0002-8148-1648 Biology, Saint Michael's College, Colchester, Vermont, UK

Competing interests: No competing interests were disclosed.

 2 8 2023 Copyright: © 2023 Avery L 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

In this manuscript, the authors aim to validate 10 commercially available moesin antibodies for western blot, immunoprecipitation, and immunofluorescence. This work makes evident that there is great variability in both what each antibody is suited for and the conditions in which it can be used. In most cases, the vendor has not included some potential uses for the antibody as validated by the authors. But in some, the vendors recommendations don’t work well for the tested application.  

Suggestions for improvement:

In introducing the work, the authors point out that moesin is implicated in inflammation seen in Alzheimer’s Disease. However, they neglect to introduce its role in other important diseases such as immunodeficiency and cancer, among many others. A more thorough introduction with a more recent literature search would make this paper searchable for many scientists who are interested in these data.

It's unclear how the antibodies were chosen for these assays. There are many commercially available moesin antibodies. It’s important to include more cited antibodies in the analysis such as those from Cell Signaling Technologies (CST) and Developmental Studies Hybridoma Bank (DSHB) as these are significantly cheaper than the antibodies currently assessed. Nonetheless, this analysis is informative for research scientists across many fields.

It should be noted that the Biotechne antibodies are available through Novus Biologicals. While Novus is a Biotechne company, the antibodies are not searchable on their site.

For immunoprecipitation data, a negative control for non-specific binding is important. The antibodies that positively IP in WT HeLa cells should be assessed in the MKO HeLa cells.

Flow cytometry is a vendor recommended application for many of these antibodies. If possible, adding this to the analysis would strengthen the results.

One concern is that one moesin antibody from Abcam (registry # AB_2885109) has since been discontinued by the manufacturer as they found that it was not specific to moesin. This antibody should be removed from analysis or addressed as nonspecific in the discussion.

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

Yes

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

Partly

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

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Immunology, T cell biology, migration, cytoskeleton, hematopoiesis

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.

Southern Kathleen McGill University, Canada

Competing interests: No competing interests were disclosed.

 3 8 2023

Thank you, Lyndsay Avery, for your response, your feedback is much appreciated.

We will be submitting a new version of the manuscript with your following suggestions included. In the introduction, we have included more recent information on Moesin’s implications in disease. As for Table 2, we have made it clear that the Bio-Techne antibodies are available from Novus Biologicals, which is a Bio-Techne brand.

As for your point regarding how the antibodies were chosen for the assays, the antibodies to be tested are donated by YCharOS partners ( https://ycharos.com/partners/), who cover nearly 28% of all antibodies listed on the Antibody registry ( antibodyregistry.org). When the industry partners are informed of upcoming targets, they donate the antibodies they have the capacity to supply at the time the study commences. At the time this study for Moesin was performed, we did not yet establish partnerships with CST nor DHSB, which is why their antibodies were not tested. It would be a great to characterize these antibodies as a follow-up study.

To respond to your comment regarding a negative control for immunoprecipitation, we too agree that negative controls to demonstrate any non-specific binding are important when validating antibodies by immunoprecipitation. That being said, under our protocol, all antibodies were tested under the same conditions within the same experiment, any antibody that did not show an enrichment in the immunoprecipitate acts as the negative control.

As majority of researchers seem to be more interested in protein localization rather than quantifying the presence of a protein in a cell, we have prioritized testing the antibodies under immunofluorescence and have included it in our standard operating procedure. Nonetheless, flow cytometry is a very useful application that will be considered as a follow-up study in the future.

To respond to your last point, Abcam made the decision to remove antibody AB_2885109 based on the result of this study and was discontinued thereafter as to provide their customers with highest quality standards of products. For more information as to why the antibody was discontinued, you can visit their website ( https://www.abcam.com/products/primary-antibodies/moesin-antibody-msn491-ab193380.html). We believe it’s important to demonstrate that our industry partners trust our validation procedure and use it as an opportunity to re-evaluate the quality of their products, which is why we kept antibody AB_2885109 in the results.

We hope you understand our reasoning for not making all of your suggested changes and are happy to receive further feedback!