Trans blot turbo transfer nitrocellulose membranes

Western blots were performed as described previously (Suzuki et al. 2015 (link)). Briefly, total proteins from cells were extracted with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Thermo Scientific, Rockford, IL). Mitochondrial fractions and cytosolic fractions were isolated using a mitochondria isolation kit for cultured cells (Thermo Scientific). Equal amounts of protein per lane (5-20 μg) were loaded onto Mini-Protean^® TGX™ gels (Biorad, Hercules, CA), transferred to Trans-Blot Turbo Transfer nitrocellulose membranes (Biorad) and probed with primary antibodies. Primary and secondary antibodies are described in supplementary data. Enhanced chemiluminescence was performed with SuperSignal West Pico (Thermo Scientific), and signal was detected by myECL imager (Thermo Scientific). Bands were quantified by densitometry using Quantity One software (Biorad). And values obtained were expressed as a ratio to each lane’s loading control value. At least three biological replicates for each experiment were assayed and representative images are shown.