The identification of high-preforming antibodies for Ubiquilin-2 for use in Western Blot, immunoprecipitation, and immunofluorescence

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.


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

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   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     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.

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