F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.131333.1 Data Note Articles The identification of high-performing antibodies for transmembrane protein 106B (TMEM106B) for use in Western blot, immunoprecipitation, and immunofluorescence

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

 Ayoubi Riham Investigation 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 Bhajiawala Ritika Writing – Original Draft Preparation https://orcid.org/0000-0003-1608-0087 2 Fanti Rebeka Writing – Original Draft Preparation 2 Prinos Panagiotis Writing – Original Draft Preparation 2 McPherson Peter S. Conceptualization Funding Acquisition Resources Supervision Writing – Original Draft Preparation 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/EDDU collaborative group ABIF Consortium Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4, Canada Structural Genomics Consortium, University of Toronto, Toronto, Quebec, M5G 1L7, 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 -ThermoFisher Scientific. The Structural Genomics Consortium receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute, the EU and EFPIA, Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.

21 3 2023 2023 12 308 9 3 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.

Transmembrane protein 106B (TMEM106B), a protein that is localized to the lysosome, is genetically linked to many neurodegenerative diseases and forms fibrils in diseased brains. The reproducibility of TMEM106B research would be enhanced if the community had access to well-characterized anti-TMEM106B antibodies. In this study, we characterized six commercially available TMEM106B antibodies for their performance 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 Q9NUM4 TMEM106B Transmembrane protein 106B antibody characterization antibody validation Western Blot immunoprecipitation immunofluorescence ALS Canada Genome Quebec OGI-210 Canadian Institutes of Health Research Foundation FDN154305 Ontario Genomics OGI-210 Mitacs fellowship ALS Association Genome Canada OGI-210 Motor Neurone Disease Association This work was supported by a grant from the Motor Neurone Disease Association (UK), The ALS Association (USA) and ALS Canada, by a Canadian Institutes of Health Research Foundation Grant (FDN154305) and by the Government of Canada through Genome Canada, Genome Quebec 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. RA and RF are supported by Mitacs fellowships. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Introduction

Transmembrane protein 106B (TMEM106B) is a genetic risk variant for many neurodegenerative diseases. The presence of the TMEM106B major risk allele, rs1990622, is suspected to be a risk factor and disease modifier for Frontotemporal Dementia (FTD), with few studies investigating its potential role in Amyotrophic Lateral Sclerosis (ALS) pathogenesis. 1 – 5

TMEM106B is a transmembrane endosomal and lysosomal glycoprotein. The protein has garnered interest lately, with the discovery that a 135 amino acid portion of the protein from its luminal C-terminal domain forms fibrils in the brains of patients with frontotemporal lobar degeneration, progressive supranuclear palsy, and dementia with Lewy bodies. 6 , 7

The roles of TMEM106B fibrils in normal lysosomal function or disease pathogenesis are not known, nor is the mechanism by which the protein is proteolyzed, or forms fibrils. 7 Mechanistic studies would be greatly facilitated with the availability of high-quality validated antibodies. Here, we compared the performance of a range of commercially available antibodies for TMEM106B and characterized several high-quality antibodies for Western blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of TMEM106B properties and function.

 Results and discussion

Our standard protocol involved comparing readouts from wild-type (WT) and knockout (KO) cells. 8 , 9 The first step was to identify a cell line(s) that expresses sufficient levels of TMEM106B to generate a measurable signal. To this end, we examined the DepMap transcriptomics databases 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 TMEM106 B at RNA levels above the average range of cancer cells analyzed. The parental and KO HAP1 cell lines 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 HZGHC005877c010 CVCL_XU38 HAP1 TMEM106B KO

For Western blot experiments, we resolved proteins from WT and TMEM106B KO cell extracts and probed them side-by-side with all antibodies in parallel ( Figure 1).

Transmembrane protein 106B antibody screening by Western Blot.

Lysates of HAP1 (WT and TMEM106B KO) were prepared and 15 μg of protein were processed for Western Blot with the indicated TMEM106B antibodies. The Ponceau stained transfers of each blot are presented are shown equal loading of WT and KO lysates and protein transfer efficiency from the acrylamide gels to the nitrocellulose membrane. Antibody dilutions were chosen according to the recommendations of the antibody supplier. Exceptions were given for antibodies 93334** and 60333-1-Ig*, which were titrated to 1/500 and 1/2000, respectively, as the signal was too weak when following the supplier’s recommendations. Antibody dilution used: ab244516 at 1/500, A20165 at 1/2000, 93334** at 1/500, 60333-1-lg* at 1/2000, PA5-34353 at 1/500, and PA5-63558 at 1/200.Predicted band size: 31 kDa. *= monoclonal antibody, **= recombinant antibody.

For immunoprecipitation experiments, we used the antibodies to immunopurify TMEM106B from HAP1 cell extracts. The performance of each antibody was evaluated by detecting the TMEM106B protein in extracts, in the immunodepleted extracts and in the immunoprecipitates ( Figure 2).

Transmembrane protein 106B antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated TMEM106B antibodies pre-coupled to Dynabeads protein G or protein A. Samples were washed and processed for Western Blot with the indicated TMEM106B antibody. For Western Blot, 93334** was used at 1/500. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=2% starting material; UB=2% unbound fraction; IP=immunoprecipitate; HC=antibody heavy chain. *= monoclonal antibody, **= recombinant antibody.

For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 10 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).

Transmembrane protein 106B antibody screening by immunofluorescence.

HAP1 WT and TMEM106B 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 TMEM106B antibodies and with the corresponding Alexa-fluor 555 coupled secondary antibody including DAPI. Acquisition of the blue (nucleus-DAPI), green (identification of WT cells), red (antibody staining) and far-red (identification of KO cells) channels were 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. When the concentration was not indicated by the supplier, we tested antibodies at 1/1000 or 1/2000. At this concentration, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab244516 at 1/100, A20165 at 1/2000, 93334** at 1/100, 60333-1-lg* at 1/2000, PA5-34353 at 1/1000, and PA5-63558 at 1/100. Bars = 10 μm. *= monoclonal antibody, **= recombinant antibody.

In conclusion, we have screened many TMEM106B commercial antibodies by Western blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions.

 Methods Antibodies

All TMEM106B antibodies are listed in Table 2 together with their corresponding Research Resource Identifiers, or RRID, to ensure the antibodies are cited properly. 11 Peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies are from Thermo Fisher Scientific (cat. number 62-6520 and 65-6120). Alexa-555 conjugated secondary goat anti-rabbit and anti-mouse antibodies are from Thermo Fisher Scientific (cat. number A21429 and A21424)

Summary of the Transmembrane protein 106B antibodies tested.
Company Catalog number Lot number RRID (Antibody Registry) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications
Abcam ab244516 GR3421643 AB_2924268 1 polyclonal - rabbit 0.10 IF
ABclonal A20165 131370201 AB_2862952 polyclonal - rabbit 1.88 Wb
Cell Signaling Technology 93334 ** 1 AB_2924267 1 recombinant-mono E7H7Z rabbit 0.10 Wb, IP, IF
Proteintech 60333-1-lg * 10002132 AB_2881442 monoclonal 5D1F8 mouse 2.00 Wb
Thermo Fisher Scientific PA5-34353 XD3572518 AB_2551705 polyclonal - rabbit 1.00 Wb
Thermo Fisher Scientific PA5-63558 XD3571635 AB_2648556 polyclonal - rabbit 0.10 IF

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

Monoclonal antibody.

Recombinant antibody.

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

 Cell culture

Both HAP1 WT and TMEM106B KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 12 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 blot experiments were performed as described in our standard operating procedure. 13 HAP1 WT and TMEM106B KO were collected in RIPA buffer (25mM Tris-HCl pH 7.6, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) from Thermo Fisher Scientific (cat number 0089901), supplemented with 1x protease inhibitor cocktail mix from 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 on nitrocellulose membranes. Proteins on the blots were visualized with Ponceau staining which is scanned to show together with individual Western blots. Blots were blocked with 5% milk for 1 h, 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 are incubated with Pierce ECL from Thermo Fisher Scientific (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. 14 Antibody-bead conjugates were prepared by adding 2 μg of antibody 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 antibodies) from Thermo Fisher Scientific (cat. number 10002D and 10004D, respectively). Tubes were rocked ~2 hours at 4°C followed by several 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) from Thermo Fisher Scientific (cat. number 87788), supplemented with protease inhibitor from 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% polyacrylamide gel. Prot-A:HRP (MilliporeSigma, cat. number P8651) was used as a secondary detection system at a dilution of 0.4 μg/mL.

 Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure. 10 HAP1 WT and TMEM106B KO were labelled with CellTracker green (Thermo Fisher Scientific, cat. number C2925) or CellTracker deep red (Thermo Fisher Scientific, cat. number C34565) fluorescence dye, respectively. 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 4% 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 three 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 containing 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 TMEM106B antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with the 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/0.45 NA air 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 Green, Alexa fluor 555 and CellTracker 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 (MetaXpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTracker 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. 15 Masks were used to generate cell outlines for intensity quantification. Figures were assembled with Adobe Illustrator.

 Data availability Underlying Data

Zenodo: Antibody Characterization Report for Transmembrane protein 106B, https://doi.org/10.5281/zenodo.7459629. 16

Zenodo: Dataset for the Transmembrane protein 106B antibody screening study, https://doi.org/10.5281/zenodo.7587647. 17

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: 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

An earlier version of the report can be found on Zenodo ( https://doi.org/10.5281/zenodo.7459629).

 References Manini A Ratti A Brusati A : TMEM106B Acts as a Modifier of Cognitive and Motor Functions in Amyotrophic Lateral Sclerosis. Int. J. Mol. Sci. 2022;23(16). 36012536 10.3390/ijms23169276 PMC9408885 Gallagher MD Suh E Grossman M : TMEM106B is a genetic modifier of frontotemporal lobar degeneration with C9orf72 hexanucleotide repeat expansions. Acta Neuropathol. 2014;127(3):407–418. 24442578 10.1007/s00401-013-1239-x PMC4003885 Hu T Chen Y Ou R : Association analysis of polymorphisms in VMAT2 and TMEM106B genes for Parkinson's disease, amyotrophic lateral sclerosis and multiple system atrophy. J. Neurol. Sci. 2017;377:65–71. 28477711 10.1016/j.jns.2017.03.028 Vass R Ashbridge E Geser F : Risk genotypes at TMEM106B are associated with cognitive impairment in amyotrophic lateral sclerosis. Acta Neuropathol. 2011;121(3):373–380. 21104415 10.1007/s00401-010-0782-y PMC3095217 Blitterswijk M van Mullen B Wojtas A : Genetic modifiers in carriers of repeat expansions in the C9ORF72 gene. Mol. Neurodegener. 2014;9:38. 25239657 10.1186/1750-1326-9-38 PMC4190282 Jiang YX Cao Q Sawaya MR : Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43. Nature. 2022;605(7909):304–309. 35344984 10.1038/s41586-022-04670-9 PMC9844993 Chang A Xiang X Wang J : Homotypic fibrillization of TMEM106B across diverse neurodegenerative diseases. Cell. 2022;185(8):1346–55.e15. 35247328 10.1016/j.cell.2022.02.026 PMC9018563 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. Bandrowski A Pairish M Eckmann P : The Antibody Registry: ten years of registering antibodies. Nucleic Acids Res. 2023;51(D1):D358–D367. 36370112 10.1093/nar/gkac927 PMC9825422 Bairoch A : The Cellosaurus, a Cell-Line Knowledge Resource. J. Biomol. Tech. 2018;29(2):25–38. 29805321 10.7171/jbt.18-2902-002 PMC5945021 Ayoubi R McPherson PS Laflamme C : Antibody Screening by Immunoblot. 2021. Ayoubi R Fotouhi M McPherson P : Antibody screening by Immunoprecitation. 2021. Stringer C Wang T Michaelos M : Cellpose: a generalist algorithm for cellular segmentation. Nat. Methods. 2021;18(1):100–106. 33318659 10.1038/s41592-020-01018-x Ayoubi R Fotouhi M Ryan J : Antibody Characterization Report for Transmembrane protein 106B. 2022. 10.5281/zenodo.7459629 Laflamme C : Dataset for the Transmembrane protein 106B antibody screening study.[Data set]. Zenodo. 2023. 10.5281/zenodo.7587647 10.5256/f1000research.144167.r178524 Reviewer response for version 1 Legname Giuseppe 1 Referee Laboratory of Prion Biology, Department of Neuroscience, Scuola Internazionale Superiore di Studi Avanzati, Trieste, Friuli-Venezia Giulia, Italy

Competing interests: No competing interests were disclosed.

 4 8 2023 Copyright: © 2023 Legname G 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 describes the various antibodies available for the study of TMEM106B. This is a useful methodological paper. The work is well organized, and the methodologies appropriately presented. I would like to suggest comparing the methods and results with those suggested by the vendors and discuss advantages to use the methodologies presented by the authors.

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?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

NA

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.

 10.5256/f1000research.144167.r183540 Reviewer response for version 1 Moritz Christian Peter 1 2 3 Referee https://orcid.org/0000-0002-9294-2323 Synaptopathies and autoantibodies (SynatAc) team, Mechanisms In Integrated Life Sciences (MELIS) Laboratory, Institute NeuroMyoGène (INMG), INSERM U1314/CNRS UMR 5284, Universités de Lyon, Université Claude Bernard Lyon 1, Lyon, France University Jean Monnet, Saint-Etienne, France Department of Neurology, University hospital of Saint-Etienne, Saint-Etienne, France

Competing interests: No competing interests were disclosed.

 4 8 2023 Copyright: © 2023 Moritz CP 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 their article, Ayoubi and colleagues are reporting about their quality tests of six different commercial anti-TMEM106B antibodies using Western blotting, immunoprecipitation, and immunocytochemistry. 

The article is interesting and useful for groups working with this protein.

Doing KO controls to test antibodies is considered the gold standard of validation tests. Nevertheless, unfortunately it is not done very often. That’s why I appreciate the work of these authors.

I would consider the following concerns.

MAJOR CONCERNS:

1) Chapter: “Results and discussion”:

In my experience it is common to describe the results in the result chapter instead of just referring to the figures.

There is no discussion in this chapter yet. The authors should interpret their results in a more concrete way. Which antibodies do they recommend for which method?

Interesting to mention in my eyes: 2 out of 6 tested antibodies don’t work in Western blotting, as they don’t bind the protein of interest, but they nonspecifically bind other proteins. One further antibody (60333-1-lg) nonspecifically binds other proteins in addition to binding the protein of interest.

In total, half of the antibodies show unspecific binding, which could harm result interpretation when using this antibody without having a KO control.

Article type "Data Note": In my perspective this article is not a "data note" in the classical sense (Journal info: "Data Notes are brief descriptions of datasets that promote the potential reuse of research data and include details of why and how the data were created; they do not include any analyses or conclusions").

The authors do not present a classical data set however. To me, an analysis and conclusions are necessary. Hence, I would ask the authors to consider another article type. Is there something like a "Technical note"?

 MINOR CONCERNS:

In their introduction, the authors speak of “a 135 amino acid portion of the protein from its luminal C-terminal domain”, that might play a pathophysiological role.

Given this information, it might be interesting which of the six antibodies have epitopes in that specific domain. I there any information available? Some ideas to address this questions methodologically ?

Results and discussion: “For Western blot experiments, we resolved proteins from WT and TMEM106B KO cell extracts and probed them side-by side with all antibodies in parallel (Figure 1).”

What means “side-by side” here? Do they come from the same membrane in order to avoid membrane-to-membrane variation? In the best case, in future experiments, the authors could even considering cutting the lanes vertically in order to even avoid lane-to-lane variation (cf. Pubmed ID 30768763).

Figure 1, panel PA5-34353:

I am a bit surprised that the “scratches” and inhomogenities in protein transfer is not equally visible in the total protein staining (ponceau). I would kindly ask the authors to check in their data files that the ponceau staining is really originating from the same membrane, because the loading control would be invalid otherwise. 

Figure 2: Why is “HC” (antibody heavy chain) only shown in the second panel? I would recommend showing it in the first panel only, or in all panels.

Figure 2: I do not understand why there is written “WB: 93334**” below each of the 6 Western blots.

Figure 3: Is the Alexa-fluor 555 not interfering with the fluorescence stainings that were applied to distinguish WT from KO?

Concluding sentence in the results section: “we have screened many TMEM106B commercial antibodies”

I recommend avoiding vague words like “many” where a more concrete indication (“six”) is possible. Same for the word “several” in the same sentence.

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?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Western blotting, antibodies, autoantibodies, ELISA, method development

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.

 10.5256/f1000research.144167.r173851 Reviewer response for version 1 Smallwood Dawn 1 Referee https://orcid.org/0000-0003-0589-1722 School of Allied Health Sciences, De Montfort University, Leicester, England, UK

Competing interests: No competing interests were disclosed.

 2 6 2023 Copyright: © 2023 Smallwood D 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

This is a useful paper, highlighting the need for antibody validation. There is important information here for TMEM106B antibody users and clear methodology for those wishing to test antibodies in their own area. Experiments are well carried out and well controlled, and figures are clear. The lack of results analysis means that figures can be interpreted by the reader for their own experimental needs, however, any misinterpretation by a reader could be avoided by adding a column to Table 2 showing the study findings alongside ‘Vendors recommended applications’.

The number of technical replicates for each method should be stated.

Full blots could be shown for immunoprecipitation results (Figure 2).

I would have found figures easier to understand with consistent naming of HAP1 WT and KO cells as HAP1 WT and HAP1 KO rather than a combination of WT and THEM106B KO (Figure 1), HAP1 (Figure 2) and WT and KO (Figure 3).

Units could be added to the markers in Figures 1 and 2.

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?

Yes

Are the protocols appropriate and is the work technically sound?

Yes

Reviewer Expertise:

Molecular biology, cell culture, medical genetics

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.