Western blot analysis

C2C12 myoblasts were seeded into 6-well plates and incubated until a confluence of 70–80%. To prepare C2C12 myotubes, cells were incubated with DMEM supplemented with 2% horse serum (Gibco, NY, USA). The differentiation medium was changed every 2 days until myotubes were formed. For Western blot analysis, myotubes or myoblasts were incubated with various concentrations of compounds or fractions for 30–60 minutes. The cells were washed with cold PBS and lysed using the lysis buffer [50 mM Tris-HCl (pH 7.6), 120 mM NaCl, 1 mM EDTA, 0.5% NP-40, 50 mM NaF] and centrifuged at 12,000 rpm for 20 minutes. The protein concentrations were determined using a protein assay kit (Bio-Rad Laboratories, Inc., CA, USA). Aliquots of lysates were electrophoresed on 8% or 12% SDS-polyacrylamide gels and then transferred electronically to polyvinylidene fluoride (PVDF) membranes (PVDF 0.45 µm, Immobilon-P, USA). Membranes were then incubated with primary antibodies for p-AMPKα Thr¹⁷², AMPKα, p-ACC Ser⁷⁹, ACC (Cell signaling Technology, Inc., Beverly, MA, USA) or mouse monoclonal actin (Abcam, Cambridge, UK). After incubation with secondary antibodies, membranes were detected using an enhanced chemiluminescence Western blot detection kit (Thermo sci., Rockford, IL, USA).