F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.131851.1 Data Note Articles The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence

[version 1; peer review: 2 approved]

 McDowell Ian Investigation Writing – Review & Editing 1 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 McPherson Peter S. Conceptualization Funding Acquisition Resources Supervision 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 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.

 30 3 2023 2023 12 355 28 2 2023 Copyright: © 2023 McDowell I 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.

Ubiquilin-2, a member of the ubiquilin protein family, plays a role in the regulation of various protein degradation pathways, and is mutated in some neurodegenerative diseases. Well-characterized anti-Ubiquilin-2 antibodies would advance reproducible research for Ubiquilin-2 and in turn, benefit the scientific community. In this study, we characterized ten Ubiquilin-2 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 Q9UHD9 UBQLN2 Ubiquilin-2 antibody characterization antibody validation Western Blot immunoprecipitation immunofluorescence ALS Society of Canada Canadian Institutes of Health Research FDN154305 Ontario Genomics OGI-210 Mitacs fellowship ALS Association Genome Canada OGI-210 Motor Neurone Disease Association Genome Quebec OGI-210 This work was supported in part by a grant from the ALS-Reproducible Antibody Platform (ALS-RAP). ALS-RAP was created as a public-private partnership by three leading ALS charities: the ALS Association (USA), the Motor Neurone Disease Association (UK), and the ALS Society of 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 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. Introduction

Ubiquilin-2, a protein encoded by the UBQLN2 gene, plays a critical role in protein degradation pathways; including the ubiquitin-proteasome system (UPS), autophagy and the endoplasmic reticulum-associated protein degradation (ERAD) pathway. 1

Disease-causing variants of UBQLN2 have been identified in patients suffering from amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). 2 These UBQLN2 mutations are predicted to be acting on ALS pathological mechanisms by causing UPS and autophagy dysfunction, 3 neuroinflammation 4 , 5 and/or formation of stress granules. 6 , 7 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 Ubiquilin-2 and validated high-quality antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of Ubiquilin-2 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 Ubiquilin-2 to generate a measurable signal. To this end, we examined the DepMap transcriptomics database to identify all cell lines that express UBQLN2 at levels greater than 2.5 log 2 (transcripts per million “TPM” +1), which we had found to be a suitable cut-off (Cancer Dependency Map Portal, RRID:SCR_017655). Commercially available HAP1 cells expressed the UBQLN2 transcript at RNA levels above the average range of cancer cells analyzed. The parental and UBQLN2 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 HZGHC004089c001 CVCL_TW09 HAP1 UBQLN2 KO

For Western Blot experiments, we resolved proteins from WT and UBQLN2 KO cell extracts and probed them side-by-side with all antibodies in parallel ( Figure 1).

Ubiquilin-2 antibody screening by Western Blot.

Lysates of HAP1 wild-type (WT) and UBQLN2 knockout (KO) were prepared, and 30 μg of protein were processed for Western Blot with the indicated Ubiquilin-2 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 according to the recommendations of the antibody supplier. When the concentration was not indicated by the supplier, which was the case for antibody 35-4400*, we tested it at 1/1000. Antibody dilution used: ab190283* at 1/2000, A9568 at 1/1000, ARP59353 at 1/500, NBP2-25164* at 1/2000, 85509** at 1/1000, PCRP-UBQLN2-1C7* at 1/26, 23449-1-AP at 1/1000, Z-UBQLN2-7** at 1/653, 35-4400* at 1/1000, and 37-7700* at 1/2000. Predicted band size: 65 kDa. *Monoclonal antibody, **Recombinant antibody.

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

Ubiquilin-2 antibody screening by immunoprecipitation.

HAP1 lysates were prepared, and IP was performed using 2.0 μg of the indicated Ubiquilin-2 antibodies pre-coupled to Dynabeads protein G or protein A or Flag-M2 magnetic beads. Samples were washed and processed for Western Blot with the indicated Ubiquilin-2 antibody. For Western Blot, 85509** was used at 1/1000. The Ponceau stained transfers of each blot are shown for similar reasons as in Figure 1. SM=4% starting material; UB=4% unbound fraction; IP=immunoprecipitate. *Monoclonal antibody, **Recombinant antibody.

For immunofluorescence experiments, 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 biases ( Figure 3).

Ubiquilin-2 antibody screening by immunofluorescence.

HAP1 wild-type (WT) and UBQLN2 knockout (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 glass bottom. Cells were stained with the indicated Ubiquilin-2 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 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. When the concentration was not indicated by the supplier, which was the case for antibodies ARP59353, 85509**, PCRP-UBQLN2-1C7* and Z-UBQLN2-7**, we tested them at 1/500, 1/200, 1/20 and 1/600, respectively. At these concentrations, the signal from each antibody was in the range of detection of the microscope used. Antibody dilution used: ab190283* at 1/1000, A9568 at 1/2000, ARP59353 at 1/500, NBP2-25164* at 1/1000, 85509** at 1/200, PCRP-UBQLN2-1C7* at 1/20, 23449-1-AP at 1/300, Z-UBQLN2-7** at 1/600, 35-4400* at 1/500, and 37-7700* at 1/500. Bars = 10 μm. *Monoclonal antibody, **Recombinant antibody.

In conclusion, we have screened Ubiquilin-2 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-performing antibodies under our standardized experimental conditions. The underlying data is uploaded to Zenodo. 16 , 17

Methods Antibodies

All Ubiquilin-2 antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (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). Peroxidase-conjugated monoclonal anti-Flag M2 is from MilliporeSigma (cat. number A8592). Alexa-555-conjugated goat anti-mouse and anti-rabbit secondary antibodies are from Thermo Fisher Scientific (cat. number A21424 and A21429). The anti-FLAG (M2 clone) conjugated with Cy3 is from MilliporeSigma (cat. number A9594).

Summary of the Ubiquilin-2 antibodies tested.
Company Catalog number Lot number RRID (Antibody Registry/Addgene) Clonality Clone ID Host Concentration (μg/μl) Vendors recommended applications
Abcam ab190283 * GR3297673-4 AB_2747782 monoclonal 6H9 mouse 1.00 Wb, IF
ABclonal A9568 92790201 AB_2772790 polyclonal - rabbit 2.42 Wb, IF
Aviva Systems Biology ARP59353 QC30343-40589 AB_10865653 polyclonal - rabbit 0.50 Wb
Bio-techne NBP2-25164 * 81919 AB_2885154 monoclonal 6H9 mouse 1.00 Wb, IF
Cell Signaling Technology 85509 ** 1 AB_2800056 recombinant-mono D7R2Z rabbit 0.12 Wb, IP
Developmental Studies Hybridoma Bank PCRP-UBQLN2-1C7 * 2021-03-04 AB_2619216 monoclonal PCRP-UBQLN2-1C7 mouse 0.03 Wb, IP
Proteintech 23449-1-AP 52217 AB_2879282 polyclonal - rabbit 0.55 Wb, IF
Structural Genomics Consortium Z-UBQLN2-7 ** YSUBQLN2A-c001 Addgene_166563 recombinant-mono YSUBQLN2A-c001 human 0.65 IP
Thermo Fisher Scientific 35-4400 * WA320043 AB_2533204 monoclonal 3B8A10 mouse 0.50 Wb, IF
Thermo Fisher Scientific 37-7700 * VA300207 AB_2533341 monoclonal 3D5E2 mouse 0.50 Wb, IP, IF

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

Monoclonal antibody.

Recombinant antibody.

 Cell culture

Both HAP1 WT and UBQLN2 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 UBQLN2 KO were collected in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Thermo Fisher Scientific, cat. number 0089901), supplemented with 1× 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× g for 15 min at 4°C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and immunoblot. BLUelf prestained protein ladder from GeneDireX (cat. number PM008-0500) was used.

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

 Antibody screening by immunoprecipitation

Immunoprecipitation experiments were performed as described in our standard operating procedure. 14 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) or anti-Flag M2 magnetic beads from MilliporeSigma (cat. number M8823). 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 (Millipore Sigma, cat. number P8340). Lysates were rocked 30 min at 4°C and spun at 110,000× g for 15 min at 4°C. 0.5 ml aliquots at 2.0 mg/ml of lysate were incubated with an antibody-bead conjugate for ~1 hr at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 ml of IP buffer and processed for SDS-PAGE and Western Blot on precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat number WXP42012BOX).

 Antibody screening by immunofluorescence

Immunofluorescence was performed as described in our standard operating procedure. 10 HAP1 WT and UBQLN2 KO were labelled with CellTracker TM green (Thermo Fisher Scientific, cat. number C2925) or CellTracker TM 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 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 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 Ubiquilin-2 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 20×/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 TM Green, Alexa fluor 555 and CellTracker TM Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns. Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition. Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference. Frequently, the focal plane varied slightly within a single field of view. To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices). Segmentation was carried out on the projections of CellTracker TM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the ‘cyto’ model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns. 15 Masks were used to generate cell outlines for intensity quantificationFigures were assembled with Adobe Illustrator.

 Data availability Underlying data

Zenodo: Antibody Characterization Report for Ubiquilin-2, https://doi.org/10.5281/zenodo.7459541. 16

Zenodo: Dataset for the Ubiquilin-2 antibody screening study, https://doi.org/10.5281/zenodo.7671135. 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, Ian McDowell, Peter S. McPherson, Chetan Raina, Wolfgang Reintsch and Kathleen Southern.

ABIF consortium: Claire M. Brown and Joel Ryan.

An earlier version of this of this article can be found on Zenodo (doi: 10.5281/zenodo.7459541).

 References Kleijnen MF Shih AH Zhou P : The hPLIC proteins may provide a link between the ubiquitination machinery and the proteasome. Mol. Cell. 2000;6(2):409–419. 10983987 10.1016/S1097-2765(00)00040-X Deng HX Chen W Hong ST : Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature. 2011;477(7363):211–215. 21857683 10.1038/nature10353 PMC3169705 Hjerpe R Bett JS Keuss MJ : UBQLN2 Mediates Autophagy-Independent Protein Aggregate Clearance by the Proteasome. Cell. 2016;166(4):935–949. 27477512 10.1016/j.cell.2016.07.001 PMC5003816 Picher-Martel V Dutta K Phaneuf D : Ubiquilin-2 drives NF-κB activity and cytosolic TDP-43 aggregation in neuronal cells. Mol. Brain. 2015;8(1):71. 26521126 10.1186/s13041-015-0162-6 PMC4628361 Picher-Martel V Renaud L Bareil C : Neuronal Expression of UBQLN2(P497H) Exacerbates TDP-43 Pathology in TDP-43(G348C) Mice through Interaction with Ubiquitin. Mol. Neurobiol. 2019;56(7):4680–4696. 30377984 10.1007/s12035-018-1411-3 PMC6647471 Renaud L Picher-Martel V Codron P : Key role of UBQLN2 in pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Acta Neuropathol. Commun. 2019;7(1):103. 31319884 10.1186/s40478-019-0758-7 PMC6889556 Dao TP Kolaitis RM Kim HJ : Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions. Mol. Cell. 2018;69(6):965–78.e6. 29526694 10.1016/j.molcel.2018.02.004 PMC6181577 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. 10.1038/s41592-020-01018-x McDowell I Fotouhi M Ryan J : Antibody Characterization Report for Ubiquilin-2. 2022. 10.5281/zenodo.7459541 Laflamme C : Dataset for the Ubiquilin-2 antibody screening study.[Dataset]. Zenodo. 2023. 10.5281/zenodo.7671135 10.5256/f1000research.144734.r173280 Reviewer response for version 1 Castaneda Carlos 1 Referee https://orcid.org/0000-0001-9634-0867 Departments of Biology and Chemistry, Syracuse University, Syracuse, New York, USA

Competing interests: No competing interests were disclosed.

 8 6 2023 Copyright: © 2023 Castaneda C 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 data note is a very important resource for the ubiquilin (UBQLN) community, as it does a very good job systematically comparing ten commercially available UBQLN2 antibodies. UBQLN2 is an important disease-linked protein whose functions are connected with the ubiquitin-proteasome system, autophagy, and stress-induced condensates. The study raises the important awareness that there are substantial differences in how these antibodies detect UBQLN2 in cell lysates, immunoprecipitation assays, and immunofluorescence experiments that examine subcellular localization of UBQLN2. The use of standardized controls and procedures enables a clear delineation as to which antibodies work best and which do not. It will be an excellent reference for many labs investigating UBQLN2 function in cells. There are only a couple of suggestions to further improve this note:

Include information (if available) on antibody epitope region; this will help ascertain whether antibodies potentially detect other related UBQLN paralogs (e.g. UBQLN1 or UBQLN4).

In the introduction, rephrase “and /or regulation of stress granules”.

The title should be reworded to say ‘high-performing’ (performing is misspelled).

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:

biophysics, protein structure and function, biomolecular condensates, protein quality control, and ubiquitin biology.

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.144734.r173282 Reviewer response for version 1 Monteiro Mervyn 1 Referee Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, MD, USA

Competing interests: No competing interests were disclosed.

 26 5 2023 Copyright: © 2023 Monteiro M 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 an excellent study comparing the utility of different commercial UBQLN2 antibodies. The authors have tested the utility and specificity of these antibodies for Western blotting, immunoprecipitation and immunofluorescence staining using a UBQLN2 expressing and knockout cell lines. Readers will be able to determine which of the antibodies are specific for UBQLN2. One limitation of the approach is the choice of the cell line, which seems to express little UBQLN4. The lack of its expression makes it difficult to assess if the UBQLN2 antibodies cross-react with UBQLN4.

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:

Neurodegenerative disorders

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.