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¹ Department of Neurology and Neurosurgery, Structural Genomics Consortium, The Montreal Neurological Institute, McGill University, Montreal, Canada

Vera Ruíz Moleón
Roles: Investigation

Charles Alende
Roles: Investigation

Maryam Fothouhi
Roles: Investigation

Sara González Bolívar
Roles: Investigation

Riham Ayoubi
Roles: Investigation, Supervision, Writing – Original Draft Preparation, Writing – Review & Editing

Vincent Francis
Roles: Writing – Original Draft Preparation, Writing – Review & Editing

Peter S McPherson
Roles: Funding Acquisition, Supervision

Carl Laflamme
Roles: Conceptualization, Funding Acquisition, Writing – Original Draft Preparation, Writing – Review & Editing

The cell lines, primary and secondary antibodies used in this study are listed in Table 1, 2, and 3, respectively. To ensure consistency with manufacturer recommendations and account for proprietary formulations (where antibody concentrations are not disclosed), antibody usage is reported as dilution ratios rather than absolute concentrations. To facilitate proper citation and unambiguous identification, all cell lines and antibodies are referenced with their corresponding Research Resource Identifiers (RRIDs).^10,11 All cell lines used in this study were regularly tested for mycoplasma contamination and were confirmed to be mycoplasma-free.

To knockdown SDCBP, HAP1 cells were treated with the SDCBP SMARTPool from Horizon Discovery (cat. Number L-008270-00) for five days. HAP1 control cells were treated with the ON-TARGETplus Non-targeting Control Pool, Horizon Discovery (cat. Number D-001810-10). Lipofectamine RNAiMAX (Thermo Fisher Scientific, cat. Number 13778030) was used to transfect the siRNA following the manufacturer’s protocol.

Lysate preparation

HAP1 ctrl and SDCBP KD 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 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 (GeneDireX, cat. Number PM008–0500) was used.

Culture medium collection

HAP1 ctrl and SDCBP KD cells were washed 3x with PBS 1x and starved for ~36 hrs (2 nights). Culture media were collected and centrifuged for 10 min at 500 x g to eliminate cells and larger contaminants, then for 10 min at 4500 x g to eliminate smaller contaminants. Culture media were then concentrated by centrifuging at 4000 x g for 30 min using the Amicon Ultra-15 Centrifugal Filter Units with a membrane NMWL of 10 kDa (MilliporeSigma cat. Number UFC901024). Culture media were finally supplemented with 1x protease inhibitor cocktail mix.

For both lysate and medium, western blots were performed with precast midi 10% Bis-Tris polyacrylamide gels (Thermo Fisher Scientific, cat. Number WG1201BOX) ran with MES SDS buffer (Thermo Fisher Scientific, cat. Number NP000202), loaded in LDS sample buffer (Thermo Fisher Scientific, cat. Number NP0008) with 1x sample reducing agent (Thermo Fisher Scientific, cat. Number NP0009) 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, membranes were incubated with the peroxidase conjugated secondary antibody diluted 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) or Clarity Western ECL Substrate (Bio-Rad, cat. Number 1705061) prior to detection with the iBright™ CL1500 Imaging System (Thermo Fisher Scientific, cat. Number A44240).

Immunoprecipitation with lysate

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 mouse antibodies) (Thermo Fisher Scientific, cat. Number 10002D and 10004D, respectively). All tubes were rocked for ~1 h at 4 °C followed by two washes to remove unbound antibodies.

HAP1 WT cells 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 x g for 15 min at 4 °C. 0.5 mL aliquots at 1.0 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 10% Bis-Tris polyacrylamide gels.

Immunoprecipitation with culture medium

Culture media from HAP1 WT were collected as described in the western blotting section above. 0.35 mL aliquots at 1.0 mg/mL of culture medium 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 10% Bis-Tris polyacrylamide gels.

HAP1 ctrl and SDCBP KD 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. Ctrl and KD 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% CO₂. Cells were fixed in 4% paraformaldehyde (PFA) (VWR, cat. Number 100503–917) in phosphate buffered saline (PBS) (Wisent, cat. Number 311–010-CL). 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 Syntenin-1 antibodies overnight at 4 °C. Cells were then washed 3 × 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 × 10 min with IF buffer and once with PBS.

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 x 0.68 microns, and a z-interval of 4 microns. 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.¹² Figures were assembled with Adobe Illustrator.

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 KO cell line suppliers, for the development of community-agreed protocols, and for their shared ideas, resources, and collaboration. Members of the group can be found below. 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: Thomas M. Durcan, Aled M. Edwards, Peter S. McPherson, Chetan Raina and Wolfgang Reintsch.

ABIF consortium: Claire M. Brown and Joel Ryan.

Thank you to the Structural Genomics Consortium, a registered charity (no. 1097737), for your support on this project. The Structural Genomics Consortium receives funding 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.

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Moleón VR, Alende C, Fothouhi M et al. A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence [version 1; peer review: 2 approved]. F1000Research 2026, 15:1021 (https://doi.org/10.12688/f1000research.183125.1)

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Current Reviewer Status: ?

VERSION 1

PUBLISHED 26 Jun 2026

Reviewer Report 04 Sep 2026

Karen Bowman, University of Leicester, Leicester, England, UK 

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Reviewer Report 22 Jul 2026

Jieya Shao, Washington University in St Louis, St. Louis, USA 

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