Fusion solo s imaging system

Prostate tissues, tumor samples or cells were lysed using 1x RIPA buffer (Cell signaling, 9806) supplemented with Phenylmethanesulfonyl fluoride (PMSF; Millipore Sigma, catalog 329-98-6) and incubated on ice for 30 min. Samples were centrifuged at 46357 g for 15 min. Protein concentration was determined by the BCA kit (Thermo Fisher 23227). Equal amounts of proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), 10% and transferred on to 0.45 mm nitrocellulose membrane (Thermo Scientific, 88018). After protein transfer, membranes were blocked in 5% milk solution and membranes were probed with the indicated antibodies overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated (HRP-linked) secondary antibodies anti-rabbit IgG (Promega, W4011, 1:5000) or anti-mouse IgG (Cell signaling, W4021, 1:5000) and developed using enhanced chemoluminescence (ECL) substrate (Thermo Scientific, 32106). Membranes were exposed to Fusion Solo S imaging system (Vilber). Blots were semi-quantitatively analyzed by densitometry using ImageJ 1.52 v (National Institutes of Health).

If not specified otherwise, cell extracts were prepared in Laemmli buffer (4% SDS, 20% glycerol, 120 mM Tris-HCl pH 6.8). Proteins were resolved by SDS–PAGE and transferred to nitrocellulose. Immunoblots were performed with appropriate antibodies and proteins visualized using the Advansta WesternBright ECL reagent and the VilberLourmat Fusion Solo S imaging system. Isolation of Triton-insoluble (chromatin-enriched) fractions was performed as previously described39 (link). Briefly, cells were rinsed twice in cold PBS, incubated for 5 min on ice in pre-extraction buffer (25 mM HEPES pH 7.4, 50 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 300 mM sucrose, 0.5% Triton X-100 and protease inhibitors). After buffer removal, adherent cellular material was harvested by scraping the cells into Laemmli buffer. The chromatin-enriched fraction was then heat denatured, sonicated and analysed by immunoblotting. Uncropped immunblots are shown in Supplementary Fig. 9.

Cells were treated as indicated and then washed with PBS. Proteins were solubilized in Laemmli buffer containing benzonase (70664–3, VWR, 1:100) added. Extracts were incubated at 37°C for 1 h. Proteins were resolved using pre-cast protein gels (Biorad, Mini-PROTEAN TGX, 4–20%, Cat# 456–1095), a Trans-Blot Turbo Transfer System (Biorad, Cat# 1704150) and Trans-Blot Turbo transfer buffer (20% EtOH). Membranes were incubated with primary antibodies overnight in 5% BSA in PBS, 0.1% Tween-20. Primary antibodies were detected using horse-radish-peroxidase (HRP) conjugated secondary antibodies (Jackson Laboratories) and the Super Signal enhanced chemiluminiscent detection kit (Thermo Scientific, Super Signal West Pico PLUS, Cat# 34580). Signals were revealed using a Fusion Solo S Imaging System (Vilber).

For EGFP-RABL2B immunoprecipitations, cells were harvested 48 h post-transfection and lysed in buffer containing 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% Igepal CA-630, and Halt protease inhibitor cocktail (#78429; Thermo Fisher Scientific). After rocking 30 min at 4°C in lysis buffer, lysates were cleared by centrifugation at 20,000g and 4°C for 10 min. For immunoprecipitation with GFP-Trap_MA beads (ChromoTek), cleared lysates were rocked for 2 h at 4°C. Beads were then eluted with Laemmli buffer and processed for SDS–PAGE in Novex Value 4–20% Tris-Glycine gels (Thermo Fisher Scientific). Proteins were then transferred to nitrocellulose or PVDF membranes and analyzed by immunoblot. For electrochemiluminescent detection, X-ray film or a Fusion Solo S imaging system (Vilber) were used. For Flag-RABL2B immunoprecipitation, cells were lysed at 4°C for 30 min in lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5% NP-40, 1 mM DTT, 0.5 mM PMSF, and 2 μg/ml leupeptin). Lysates were then cleared by centrifugation (17,000g for 10 min) and their protein content analyzed. Immunoprecipitations were performed on 2 mg of total protein by incubating 2 h at 4°C with anti-Flag agarose beads (Sigma-Aldrich). Beads were then washed in lysis buffer and bound polypeptides analyzed by SDS–PAGE and immunoblotting. 20 μg of lysate was loaded in the input lane.

Cells were lysed with a lysis buffer (PBS buffer supplemented with 0.2 mM PMSF, 2% Triton-X, and 1% phosphatase inhibitor cocktail II (Sigma-Aldrich, St. Louis, MO, USA)). Protein concentrations were determined using the Bradford assay, and equal amounts of protein (20 µg/well) were resolved via SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk in TTBS solution (0.5% TBS-Tween 20) for 1 h, membranes were incubated overnight at 4 °C with primary antibodies (Table S3). The next day, membranes were washed with TTBS six times and treated with a secondary antibody (1: 10,000 dilution) at room temperature (RT) for 1 h. Proteins were visualized using a chemiluminescent Femto reagent (Thermo Fisher Scientific) and a Fusion Solo S imaging system (Vilber Lourmat, Paris, France). Western blots were quantified using Evolution-Capt v18.10 software supplied by Vilber Lourmat (Paris, France).