Identification of high-performing antibodies for Vacuolar protein sorting-associated protein 35 (hVPS35) for use in Western Blot, immunoprecipitation and immunofluorescence

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


Introduction
Vacuolar protein sorting-associated protein 35 (hVPS35) is a component of the retromer complex. 1 The retromer complex is a multimeric protein complex responsible for sorting transmembrane cargo from the endosome to the trans-Golgi network or plasma membrane, a pathway which is conserved across all eukaryotes. 1,2Composed of two major subcomplexes, the cargo-selective complex and the membrane bound vacuolar protein sorting-associated protein (VPS) complex, hVPS35 serves as an essential component of the VPS complex where it mediates the recruitment of the retromer complex to the endosomal membrane. 18][9] A missense mutation in the gene, D620N, has been reported in numerous individuals and families with PD. [10][11][12][13][14][15][16] Further research is required to investigate the molecular mechanisms in which VPS35 mutations induces neurodegeneration in PD. 7 Mechanistic studies would be greatly facilitated with the availability of high-performing antibodies.Under our standardized procedure, a high-performing antibody, or a successful antibody, can be defined according to its application.In Western blot, a high-performing antibody will specifically immunodetects the target protein in the Wild-type (WT) but not in the knockout (KO) lysate.In immunoprecipitation, a high-performing antibody immunocaptures the target protein to at least 10% of the starting material.For immunofluorescence, a high-performing antibody immunolocalizes the target protein by generating a fluorescent signal that is 1.5-fold higher in WT cells than in the KO cells. 17re, we compared the performance of a range of commercially-available antibodies for hVPS35 and identified highperforming antibodies for Western Blot, immunoprecipitation and immunofluorescence, enabling biochemical and cellular assessment of hVPS35 properties and function.

Results and discussion
8][19][20][21][22][23] The first step was to identify a cell line(s) that expresses sufficient levels of hVPS35 to generate a measurable signal.To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID: SCR_017655).Commercially available HAP1 cells expressed the hVPS35 transcript at RNA levels above the average range of cancer cells analyzed.Parental and VPS35 KO HAP1 cells were obtained from Horizon Discovery (Table 1).

REVISED Amendments from Version 1
In the introduction section of this revised article, we have incorporated additional comprehensive information about PARK17, along with an explanation of what a high-performing antibody is in each tested application.To the results and discussion section, we have included a description as to why the authors and YCharOS initiative does not score nor interpret antibody characterization data.
Any further responses from the reviewers can be found at the end of the article For immunofluorescence, as described previously, antibodies were screened using a mosaic strategy. 24In 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).
In conclusion, we have screened hVPS35 commercial antibodies by Western Blot, immunoprecipitation and immunofluorescence and identified several high-quality antibodies under our standardized experimental conditions.Under our standardized experimental conditions, several high-quality antibodies were identified, however, the authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocolsany conclusions remain relevant within the confines of the experimental setup and cell line used in this study.Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles.This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.
The underlying data can be found of Zenodo. 25,26

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

Cell culture
Both HAP1 WT and VPS35 KO cell lines used are listed in Table 1, together with their corresponding RRID, to ensure the cell lines are cited properly. 28 Western Blots were performed with pre-cast mini 4-15% gradient polyacrylamide gels from Bio-Rad (cat.number 4561084) and transferred onto nitrocellulose membranes.Proteins on the blots were visualized with Ponceau S staining (Thermo Fisher Scientific, cat.number BP103-10) which is scanned to show together with individual Western Blot.Blots were blocked with 5% milk for 1 hr, and antibodies were incubated overnight at 4°C with 5% bovine serum albumin (BSA) (Wisent, cat.number 800-095) in TBS with 0,1% Tween 20 (TBST) (Cell Signaling Technology, cat.number 9997).Following three washes with TBST, the peroxidase conjugated secondary antibody was incubated at a dilution of ~0.2 μg/mL in TBST with 5% milk for 1 hr at room temperature followed by three washes with TBST.Membranes were incubated with Pierce ECL from Thermo Fisher Scientific (cat.number 32106) prior to detection with the HyBlot CL autoradiography films from Denville (cat.number 1159T41).

Antibody screening by immunoprecipitation
Immunoprecipitation was performed as described in our standard operating procedure. 30Antibody-bead conjugates were prepared by adding 2 μg to 500 μL of Pierce IP Lysis Buffer from Thermo Fisher Scientific (cat.number 87788) in a 1.5 mL microcentrifuge tube, together with 30μL of Dynabeads protein A-(for rabbit antibodies) or protein G-(for mouse antibodies) from Thermo Fisher Scientific (cat.number 10002D and 10004D, respectively).Tubes were rocked for ~1 hr at 4°C followed by two washes to remove unbound antibodies.HAP1 WT were collected in Pierce IP buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific, cat.number 87788) supplemented with protease inhibitor (MilliporeSigma, cat.number P8340).Lysates were rocked for 30 min at 4°C and spun at 110,000 x g for 15 min at 4°C. 0.5 mL aliquots at 2.0 mg/mL of lysate were incubated with an antibody-bead conjugate for ~2 hrs at 4°C.The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP lysis buffer and processed for SDS-PAGE and Western Blot on a pre-cast mini 4-15% gradient polyacrylamide gels.Prot-A: HRP (MilliporeSigma, cat.number P8651) was used as a secondary detection system at a concentration of 0.3 μg/mL.0][21][22][23][24] HAP1 WT and VPS35 KO were labelled with a green and a far-red fluorescence dye, respectively.The fluorescent dyes used are from Thermo Fisher Scientific (cat.number C2925 and C34565).The nuclei were labelled with DAPI (Thermo Fisher Scientific, cat.Number D3571) fluorescent stain.WT and KO cells were plated in 96 well glass plates (Perkin Elmer, cat.number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37 o C, 5% CO 2 .Cells were fixed in paraformaldehyde (PFA) (Beantown chemical, cat.number 140770-10ml) in phosphate buffered saline (PBS) (Wisent, cat.number 311-010-CL) for 15 min at room temperature and then washed 3 times with PBS.Cells were permeabilized in PBS with 0,1% Triton X-100 (Thermo Fisher Scientific, cat.number BP151-500) for 10 min at room temperature and blocked with PBS with 5% BSA, 5% goat serum (Gibco, cat.number 16210-064) and 0.01% Triton X-100 for 30 min at room temperature.Cells were incubated with IF buffer (PBS, 5% BSA, 0,01% Triton X-100) containing the primary hVPS35 antibodies overnight at 4°C.Cells were then washed 3 Â 10 min with IF buffer and incubated with corresponding Alexa Fluor 555-conjugated secondary antibodies in IF buffer at a dilution of 1.0 μg/mL for 1 hr at room temperature with DAPI.Cells were washed 3 Â 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/0.95NA water objective lens and scientific CMOS camera (16-bit, 1.97mm field of view), equipped with 395, 475, 555 and 635 nm solid state LED lights (Lumencor Aura III light engine) and bandpass emission filters (432/36 nm, 520/35 nm, 600/37 nm and 692/40 nm) to excite and capture fluorescence emission for DAPI, CellTracker TM Green, Alexa fluor 555 and CellTracker TM Red, respectively.Images had pixel sizes of 0.68 x 0.68 microns.Exposure time was set with maximal (relevant) pixel intensity ~80% of dynamic range and verified on multiple wells before acquisition.Since the IF staining varied depending on the primary antibody used, the exposure time was set using the most intensely stained well as reference.Frequently, the focal plane varied slightly within a single field of view.To remedy this issue, a stack of three images per channel was acquired at a z-interval of 4 microns per field and best focus projections were generated during the acquisition (MetaExpress v6.7.1, Molecular Devices).Segmentation was carried out on the projections of CellTracker TM channels using CellPose v1.0 on green (WT) and far-red (KO) channels, using as parameters the 'cyto' model to detect whole cells, and using an estimated diameter tested for each cell type, between 15 and 20 microns. 31

Emily Sontag
Marquette University, Milwaukee, Wisconsin, USA The article characterizes 13 different commercial antibodies to the human VPS35 protein, which is associated with Parkinson's disease.This information is highly valuable to the field, but a few clarifications will assist readers in interpreting the data and using it to make decisions on which reagents to use in their own experiments.
The amendments provide additional information about PARK17, but introduce a bit of confusion.
In the third sentence of the first paragraph of the introduction, it is unclear if the retromer complex or the hVPS35 is composed of 2 subcomplexes.Additionally, the first sentence of the second paragraph of the introduction is confusing as it appears that the sentence is repeated within itself.The definition of a high-performance for each application is very clearly stated.
While the authors are clear on why they do not offer analyses or recommendations, it would be very helpful to include quantification of the Western blots from the IP experiments and the intensity values of the immunofluorescence.Several of the antibodies do not appear to meet the threshold for high-performance by those techniques (10% of input and a 1.5-fold increase in signal, respectively).The measured values would aid experts in interpreting the data and determining which antibodies meet the standard of a high-performing antibody.
Additionally, it is unclear why the GTX635821 and 81453 antibodies were chosen for the Western blot analysis of the IP experiments.The NBP2-75710 antibody appears to give the best signal on the Western blot screening in Figure 1.The legend for Figure 3 states that the representative images of the blue and red channels are merged, but they are shown as separate grayscale images.
The article is scientifically sound and contributes to the field, but could be improved with minor revisions.
Is the rationale for creating the dataset(s) clearly described?

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?Partly Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Biochemistry, Cell Biology, protein homeostasis, ESCRT and VPS protein function.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The article is a data note that evaluates different commercial antibodies for the human Vacuolar protein sorting-associated protein 35 (hVPS35),a.subunit of the Retromer complex, also composed of VPS26 and VPS29 subunits.Retromer is involved in several cargo sorting processes at the endosomes, regulating endosome to plasma membrane trafficking of cargoes and the endosome to Golgi apparatus retrograde transport of cargoes such as the mannose-6-phosphate receptors.Mutations in Retromer are associated with neurodegenerative conditions, such as Parkinson's Disease (PD).This article is particularly relevant as a data note for evaluating different commercial antibodies for VPS35 detection, but some mistakes should be addressed.
In the Abstract and the Introduction, it is mentioned a type 17 Parkinson's disease progression.
The VPS35 gene is the PARK17, but there are not 17 types of PD.The authors could check the PARK genes that historically are the genes that have been linked to PD. Reference 1 can be complemented with other articles; I suggest including the articles: Is the rationale for creating the dataset(s) clearly described?
The study is relevant to the field.It is necessary to characterize and find reliable antibodies that can detect the VPS35, without false positives, for describing and ascribing different and eventually new retromer functions and cargoes.The authors established that the main AIM of the data note is to use it as a guide to select the appropriate antibody for the research.The guide could help scientists in the field to invest in quality antibodies, saving time, money, and other resources in the characterization of antibodies that, in the end, will be useless.This AIM is accomplished; as a guide, this article gives useful information.However, it could be improved by discussing the reliability of the antibodies tested and the pertinence of the results obtained.

Are the protocols appropriate, and is the work technically sound?
The authors analyze the antibody's performance in three techniques widely utilized in cell biology: western blot, immunoprecipitation, and immunofluorescence.These are techniques often used in the field, including membrane trafficking studies and neurobiology studies.Nevertheless, the results could be improved.
The authors decided to test the antibodies in the carcinogenic cell line HAP1 should explore other non-carcinogenic cell lines as well as neuronal cell lines considering that many mutants of VPS35 are associated with neurodegenerative diseases, including PD.Moreover, even among cells of the same species, antibody performance also depends on the cellular type; therefore, it would be desirable to test the antibodies in a neuron-like cell line as H4, SH-Sy5Y, to mention some.
In the western blot experiments, there are unspecific bands in the WT and KO lines for GTX116260, PA5-30654, and ab57632*.This should be remarked in an additional table or Table 2 for scientists to evaluate the pertinence of these antibodies.
In the immunofluorescence, the signal shown does not correlate with an expected signal of Retromer following the VPS35 subunit.It should show a vesicular pattern with identifiable structures.This could be correlated with other endosome markers or cargos such as mannose-6phosphate, the GTPase Rab7, or the expression of a GFP-tag VPS35.Besides, authors should consider testing other fixations as methanol and performing negative controls in nonpermeabilized cells.I wonder if 20X is the ideal magnification for the analysis because the label is very diffused.However, comparing the WT and KO lines in the same field is a great idea.

Are sufficient details of methods and materials provided to allow replication by others?
The methods are clear and detailed.However, the authors could explain why, for the western blots, they decided to block with milk and then incubate the primary antibody in BSA.For example.for some antibodies, the BSA is also required for blocking.A clarification on the concept of highperforming antibodies and what it entails should be included.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
Author Response 06 Oct 2023

Kathleen Southern
Thank you for your review and feedback.We will be addressing your comments and submitting a new version of the article, with many of your suggestions included.
To the abstract and introduction, we have removed the mention of type 17 Parkinson's disease.Furthermore, in the introduction we have included information to mention other PARK genes that have been identified in research, including PARK17.We are always looking to improve on our articles and hope this satisfies your feedback!
In regard to your comment on the rationale for creating the dataset, we understand that you believe it would enhance the quality of the article if we discussed the performance of the antibodies tested and reported their specificity or selectivity in regards to the results.In response to this feedback, it's important to clarify that YCharOs does not engage in result analysis nor do we offer explicit antibody recommendations.We're not experts of these proteins and prefer to remain impartial which is why we carry out the study without interpreting the results.The purpose of YCharOS is to deliver antibody characterization reports as a public good collection to benefit biomedical research as a whole.It is for this reason that we chose to publish our articles using the Data Note format, as it does not require result analysis but rather presents KO characterization data for experts of the individual targets to further their studies and projects.We have found that, for the most part, scientists viewing our articles have the expertise to the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.We understand that this point has not been made clear within the article, which is why we have added a new paragraph to the Results and Discussion section, explaining our reasonings as to why we do not recommend or score the antibodies tested.
In terms of cell lines, at this point, testing and validating the antibodies in various neuronal cell lines is outside the scope of our standard protocol and study.We utilize the Cancer Dependency Map (DepMap) as a guide to select a cell line with adequate levels of protein of interest.That being said, we hope to revisit all Parkinson's disease-related protein targets in the future by quantifying the antibody performance on a larger scale and analyzing protein expression in various neuronal cell lines.
In terms of evaluating the antibodies you mentioned in Western blot, as previously mentioned, we believe scientists viewing the article will have the expertise to see that the antibodies mentioned are non-selective using our current experimental set-up.
The reason as to why different fixation methods were not used, is because with our current workflow we identified antibodies that specifically target hVPS35 by immunofluorescence The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Table 1 .
Summary of the cell lines used.
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.

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