Western Blot Analysis of Myoblast Differentiation

L6 myoblasts were cultured at 90% confluence and differentiated. Myoblasts and differentiated myotubes were homogenized with lysis buffer (25 mM Tris, pH 7.9, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA, 5% glycereol, protease inhibitors). Protein concentration of cell lysates was determined by the Protein Assay Kit (iNtRON biotechnology, Korea), an equal amount of cells lysates was separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare, Germany). The membranes were incubated with specific antibodies against MHC, p-AKT, AKT, p-ERK, ERK, and GAPDH. The blots were then incubated with horseradish peroxidase conjugated anti-mouse secondary antibodies or horseradish peroxidase conjugated anti-rabbit secondary antibodies. Bands were detected by the ECL detection system (GE Healthcare, Germany) and band intensities were quantified by ImageJ software (Yi, Hwang, Oh, Kim, Ryu, et al. 2018 (link)).