Identification of high-performing antibodies for Moesin for use in Western Blot, immunoprecipitation, and immunofluorescence

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 have characterized ten Moesin commercial antibodies in 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.


Introduction
Moesin is a cytoskeletal adaptor protein that belongs to the Ezrin-Radixin-Moesin (ERM) family of proteins which connects the actin cytoskeleton to the plasma membrane, regulating the structure and function of specific domains of the cell cortex. 1,2Moesin plays a pertinent role in immunity, acting on T and B-cell homeostasis and self-tolerance. 3,4As such, specific mutations in this ERM protein have implications in immunodeficiency. 5,69][10][11] Proteomic and protein co-expression network analysis of Alzheimer's Disease (AD) brain has revealed a module that is enriched in inflammation-related proteins. 12Moesin, 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. 13Mechanistic 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 in Western Blot, immunoprecipitation and immunofluorescence using a knockout based approach.This article serves as a guide to help researchers select high-quality antibodies for their specific needs, facilitating the biochemical and cellular assessment of Moesin properties and function.

REVISED Amendments from Version 2
This version of the article includes a clarification of how this Data Note it is part of a larger collaborative initiative seeking to address the antibody liability crisis by characterizing commercial antibodies for human proteins and making the corresponding data open and accessible.
Furthermore, errors in figure legends 1 and 3 minor errors by a reviewer were addressed, including the removal of the second star for monoclonal antibody ab193380.

Results and discussion
Our standard protocol involves comparing readouts from wild-type and knockout cells. 14,15The 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).
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).
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).
For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 16First, HeLA WT and MSN KO cell lines were labelled with different coloured fluorescent dyes to distinguish the two cell lines, and then the ten Moesin antibodies were evaluated.Cells were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 3).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.

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).

Cell culture
HeLa WT and MSN KO cells used are listed in Table 1

Antibody screening by Western Blot
Western Blots were performed as described in our standard operating procedure. 17HeLa 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 Â 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. 18Antibody-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 Â 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. 16HeLa 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 40Â 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.
-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). 19ta 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.
An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.4724169). 19

Figures 1 and 3:
Regarding the NBP2-32876, NBP2-44579, NBP2-44580 antibodies (Bio-Techne), the authors used U2OS cells for the same western blot and immunofluorescence images in the company's webpage, while they described using HeLa cells in this manuscript.2?
Figure 3: Is there any reason why the vendor does not recommend IF for ab193380 in Table 2?
Minor comments: Figure 3: yellow and magenta dashed "line" should be yellow and magenta dashed "lines".
Please carefully check citations, figure labels, format, language, and readability once more.
I would like to thank the authors for their efforts to promote open science.

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? Yes
Are the datasets clearly presented in a useable and accessible format?
"yellow and magenta dashed line" has been changed to "yellow and magenta dashed lines" Thank you for being observant and catching these errors!A new version of this publication has been submitted to F1000 which include the changes above.
Please see our responses below regarding your major comments: Figure 1: Regarding the ab52490 antibody, the author loaded 20 ug of protein for western blot in the company's webpage, while they described 25 ug in this manuscript.
When performing the Western Blot, each lane of the gel is loaded with equal amounts of protein.That is why we couldn't make an exception for this specific antibody as we have to have equal amounts of protein in order to correctly compare the antibodies performance side by side.

Figures 1 and 3:
Regarding the NBP2-32876, NBP2-44579, NBP2-44580 antibodies (Bio-Techne), the authors used U2OS cells for the same western blot and immunofluorescence images in the company's webpage, while they described using HeLa cells in this manuscript.
As described in our results & discussion section, we select cell lines from based on public transcriptomic database, DepMap.The cell lines chosen are based on transcript level cut-off of the protein of interest.Based on the expression datasheet for Moesin, available on DepMap, HeLa cells expressed higher RNA levels of MSN compared to U2OS, which is why HeLa WT and MSN KO cells were obtained.2?
The vendors recommend applications column is prior to validation by YCharOS.Once the validation data for Wb, IP and IF is collected, it is shared with the antibody manufacturer partners.Based on the results, it is then up the discretion of each companies whether or not they change the vendor recommended applications on their website.If you were to visit the vendor's website for antibodies MA5-17130 and NBP2-32876, you'd see that they have included IP in their recommended applications based on the characterization data provided by YCharOS.Antibody ab52490, although was not successful in IP was not removed by the supplier.

Figure 3: Is there any reason why the vendor does not recommend IF for ab193380 in Table 2?
As mentioned previously, the list of vendor recommended applications in Table 2 is based on the vendors website, prior to the availability of validation data provided by YCharOS.The vendors did not know prior to YCharOS testing that this antibody was successful in IF.
Regardless, the vendors have decided to discontinue this specific antibody from their catalog.
Thank you again for this thorough and detailed report.We hope we addressed all of your concerns and that the article meets your standards to be approved.

Competing Interests:
No competing interests were disclosed.

Version 1
Reviewer Report 02 August 2023 https://doi.org/10.5256/f1000research.142858.r177143 © 2023 Na S. This is an open access peer review report 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.

Sungsoo Na
Indiana University Purdue University Indianapolis, Indianapolis, Indiana, USA 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".

Lyndsay Avery
Biology, Saint Michael's College, Colchester, Vermont, UK 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.

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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.

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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.

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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.

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

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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.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.

Figure 2 .
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.

Figure 2 :
Figure 2: Is there any reason why the vendor recommends IP for ab52490, and does not recommend for MA5-17130 and NBP2-32876 in Table2?

Figure 2 :
Figure 2: Is there any reason why the vendor recommends IP for ab52490, and does not

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Is the rationale for creating the dataset(s) clearly described?PartlyAre the protocols appropriate and is the work technically sound?YesAre sufficient details of methods and materials provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

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? Partly Competing Interests:
No competing interests were disclosed.

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.
This is an open access peer review report 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.