Pierce lane marker non reducing sample buffer

For protein extraction, cells were washed twice with ice-cooled 1xPBS before lysis. Ice-cooled lysis buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, supplemented with EDTA-free protease inhibitor (Roche Applied Science) and PhosSTOP phosphatase inhibitor (Roche Applied Science) was added to cells. Cells were scraped off and sonicated on ice. Protein lysates were centrifuged for 5 min in 10,000 × g at 4 °C. Supernatant was collected and quantified by DC Protein Assay Kit (Bio-Rad). The lysate was diluted to 1 μg/μL by using lysis buffer. The diluted lysates were mixed with 5x sampling buffer (300 mM Tris-HCl pH6.8, 50% glycerol, 10%(v/v) β-mercaptoethanol, 10%(w/v) SDS and 50 mg bromophenol blue) or Pierce Lane Marker Non-Reducing Sample Buffer (Thermo Scientific) in a 1-to-4 ratio for reducing or non-reducing Western blot analysis, respectively. For non-reducing analysis, gels were soaked with 25 mM DTT for 10 min before electrotransfer to Immobilon-P PVDF membrane (Sigma-Aldrich).

Western blot analysis was performed following our published protocol (11 (link)) with minor modification. Briefly, cells were lysed with the Pierce RIPA lysis buffer (89901; Thermo Fisher Scientific) containing Halt Protease Inhibitor Cocktail (87785; Thermo Fisher Scientific). Total protein concentration was determined using the DC Protein Assay Kit (5000111; Bio-Rad). The cell lysates were solubilized in Pierce Lane Marker Non-Reducing Sample Buffer (39001; Thermo Fisher Scientific) and heated at 95°C for 10 min after SDS–PAGE and Western blotting. The information for the antibodies used are available in Table S3. Specific protein bands on Western blots were quantified by the ImageJ 64 software (https://imagej.nih.gov/ij/) using Gel analyzer script. Densitometry data were normalized to loading control (GAPDH or β-actin) and then to a basal condition (e.g., vehicle and/or scrambled sequence siRNA).

Table S3 Antibodies for Western blotting.