Inherent limitations are associated with the antibody characterization platform used in this study. First, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all available antibodies against any given targets. While YCharOS partners provide access to the majority of renewable antibodies, some widely used polyclonal antibodies may not be included.

Second, the YCharOS approach is protein-agnostic and aims to provide objective data on antibody performance without predefined expectations regarding molecular weight or localization. As such, only a brief overview of the protein target is provided, and expert interpretation of banding patterns and subcellular localization is encouraged.

Third, experiments are not performed in biological replicates due to the parallel testing of multiple antibodies recognizing distinct epitopes. The identification of at least one specific antibody supports target expression in WT cells and its absence in KO cells, providing a reference for evaluating other antibodies. Experiments are performed using standardized conditions and master mixes to minimize variability. In immunofluorescence, testing at multiple antibody concentrations provides an additional assessment of specificity. Experiments may be repeated when no signal is detected. Furthermore, these data are generated independently of manufacturers’ validation processes, effectively constituting an external replication.

Finally, conclusions are limited to the experimental conditions and cell line used. The reliance on a single cell type represents a constraint, as protein expression levels can influence antibody performance. The use of cancer cell lines also introduces potential confounders, including mutations that may affect antibody-epitope binding. These limitations are inherent to cell line-based validation approaches.

The cell lines used in this study are listed in Table 1. To facilitate proper citation and unambiguous identification, all cell lines and antibodies are referenced with their corresponding Research Resource Identifiers (RRIDs). ⁸ All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free.

Primary antibodies are listed in Table 2 with RRIDs. ⁹ Working dilutions for western blot and immunofluorescence, and volumes corresponding to 2 μg input for immunoprecipitation, are provided in Table 3. Secondary antibodies and their working concentrations are listed in Table 4. Antibody usage is reported as dilution ratios.

MCF7 WT and SCARB2 KO cells 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 89901) supplemented with 1x protease inhibitor cocktail mix (MilliporeSigma, cat. number P8340). Lysates were sonicated briefly and incubated 30 min on ice. Lysates were spun at ~110,000 × g for 15 min at 4°C and 30 μg of protein aliquots of the supernatants were analyzed by SDS-PAGE and western blot. BLUelf prestained protein ladder (GeneDireX, cat. number PM008-0500) was used.

Western blots were performed with precast midi 4-20% Tris-Glycine polyacrylamide gels (Thermo Fisher Scientific, cat. number WXP42012BOX) ran with Tris/Glycine/SDS buffer (Bio-Rad, cat. number 1610772), loaded in Laemmli loading sample buffer (Thermo Fisher Scientific, cat. number AAJ61337AD) and transferred on 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 O/N at 4°C with 5% milk 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-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 microcentrifuge tube, together with 30 μl of Dynabeads protein A- (for rabbit antibodies) or protein G- (for rat antibodies) (Thermo Fisher Scientific, cat. number 10002D and 10004D, respectively). Tubes were rocked for ~1 h at 4°C followed by two washes to remove unbound antibodies.

MCF7 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) supplemented with protease inhibitor. 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 1 mg/ml of lysate were incubated with an antibody-bead conjugate for ~1 h 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.

MCF7 WT and SCARB2 KO cells were labelled with a green and a far-red fluorescence dye, respectively (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 on 96-well plate with optically clear flat-bottom (Perkin Elmer, cat. number 6055300) as a mosaic and incubated for 24 hrs in a cell culture incubator at 37°C, 5% CO ₂. Culture medium was removed, and cells were fixed in 4% paraformaldehyde (PFA) (VWR, cat. number 100503-917) in phosphate buffered saline (PBS) (Wisent, cat. number 311-010-CL) for 10 min at room temperature. Cells were permeabilized in PBS1x with 0.05% Saponin (MilliporeSigma, cat. number 47036) or 0.1% Triton X-100 (Thermo Fisher Scientific, cat. number BP151-500) for 10 min at room temperature. Cells were blocked in IF buffer (PBS1x with 5% BSA and 0.005% Saponin or 0.01% Triton X-100) with 5% goat serum (Gibco, cat. number 16210-064) for 1 h at room temperature. Cells were incubated with the corresponding IF buffer containing the primary SCARB2 antibodies overnight at 4°C. Cells were then washed 3 × 10 min with IF buffer and incubated with the corresponding Fluor 555-conjugated secondary antibody in IF buffer for 1 hr at room temperature. Cells were washed 3 × 10 min with PBS1x then incubated with DAPI and washed once with PBS1x.

Images were acquired on an ImageXpress micro confocal high-content microscopy system (Molecular Devices), using a 20x NA 0.94 air objective and scientific CMOS cameras, equipped with 395, 475, 555 and 635 nm solid state LED lights (lumencor Aura III light engine) and bandpass filters to excite DAPI, Cellmask Green, Alexa-555 and Cellmask Red, respectively. Images had pixel sizes of 0.68 × 0.68 microns, and a z-interval of 4 microns. A minimum of 500 WT and KO cells were imaged per antibody. For analysis and visualization, shading correction (shade only) was carried out for all images. Then, maximum intensity projections were generated using 3 z-slices. Segmentation was carried out separately on maximum intensity projections of Cellmask channels using CellPose 1.0, and masks were used to generate outlines and for intensity quantification. ¹⁰ We have developed a collection of scripts in Python and in ImageJ/FIJII made openly available on GitHub ( https://github.com/ABIF-McGill/YCharOS_IF_characterization ). ⁴ Figures were assembled with Adobe Illustrator.