Clarity wb ecl substrate

For next experiments all antibodies details are described in Supplementary Table S4.
Briefly, cells were scraped in lysis Buffer (Kinexus Bioinformatics Corporation, Vancouver, British Columbia, Canada) on ice. Protein quantification was performed using the Pierce BCA Protein Kit (Thermo Scientific Inc.), according to the manufacturer’s instructions. Western blot (WB) was performed as previously described^{53 (link)} using specific primary antibodies. After primary antibody incubation overnight at 4 °C, specific conjugated horseradish peroxidase secondary antibodies (Bio-Rad, USA) were incubated 1 h R/T. Chemiluminescence was detected with Clarity WB ECL substrate (Bio-Rad, USA) and evaluated using ImageJ software (version 1.6.1, from National Institutes of Health). β-actin served as control of the total loaded protein.

Twenty µg or 3 µg of total protein or histones, respectively, were separated in Mini-protean TGX Stain-Free gels 4-15% (Bio-Rad, Hercules, California, USA) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, were transferred by using a Trans-Blot Turbo transfer system (Bio-Rad) into an immunoblot nitrocellulose membrane in a transfer buffer containing ethanol, distilled water, and Trans-Blot® TurboTM 5x Tranfer Buffer (Bio-Rad). After that, membranes were blocked with 5% milk in tris-buffer saline (TBS)/0.1% Tween (TBS-T) for 1 h at RT. Then, membranes were incubated with primary antibodies overnight at 4°C (Supplementary Table S2). Next, membranes were washed with TBS-T and incubated with a secondary antibody coupled with horseradish peroxidase (Supplementary Table S2) for 1 h at RT. Blots were visualized by chemiluminescence (Clarity WB ECL substrate, Bio-Rad) using a ChemiDoc XRS+ (Bio-Rad). Western blots were quantified using band densitometry analysis with ImageJ software (version 1.6.1., National Institute of Health). All antibodies and dilutions used are described in Supplementary Table S2.

SVHUC1, UMUC3^KD and UMUC3^CTRL cell lines were grown until 80% confluence and homogenized in Kinexus lysis buffer supplemented with proteases inhibitors cocktail. Then, cells were sonicated for 5 cycles of 30 seconds ON and 30 seconds OFF (Bioruptorâ, Diagenode, Liège, Belgium). After centrifugation, the supernatant was collected, and total protein was quantified according to the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific Inc.), according to the manufacture procedure.
Thirty µg total protein were separated in 10% polyacrylamide gel by SDS-PAGE and transferred onto an immunoblot PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA) in a 25mM Tris-base/ glycine buffer using a Trans-Blot Turbo Transfer system (Bio-Rad Laboratories). Membranes were blocked with 5% bovine serum albumin (BSA) in TBS/0.1% Tween (TBS/T pH=7.6) for 2h at RT. After incubation with VIM primary antibody (NCL-L-VIM-V9, Leica), membranes were washed in TBS/T and incubated with secondary antibody coupled with horseradish peroxidase, for 1h at RT. Binding was visualized by chemiluminescence (Clarity WB ECL substrate, Bio-Rad) and quantification was performed using band densitometry analysis from the ImageJ software (version 1.6.1, National Institutes of Health). β-Actin (A1978, Sigma-Aldrich) was used as loading control.