Immunoblotting of Differentiated Myotubes

Immunoblotting was performed as described previously^{19 (link)}. Briefly, differentiated myotubes were lysed in RIPA buffer (10 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, pH 7.5) with cell scraper. Following centrifugation (16,000 g for 15 min at 4°C), supernatants were collected for Bradford assay to quantify total protein concentrations. Following SDS-PAGE, samples were transferred to the PVDF membrane. Subsequently, the membranes were incubated in TBST containing 5% nonfat dry milk for 30 min prior to overnight incubation with respective primary antibodies (rabbit polyclonal anti-VDAC1/porin [Abcam] and mouse monoclonal α-tubulin [Sigma-Aldrich]). After washing twice in TBST, the membranes were further incubated with HRP-conjugated secondary antibodies for 40 min, followed by washing thrice with TBST. Then, membranes were subjected to standard enhanced chemiluminescence (Thermo Fisher Scientific) method for visualization. The resulting band intensities were quantified by ImageJ software (NIH).