2.11. Preparation of Protein Lysates and Western Blot Analysis

After the experiments were completed, the C2C12 myotubes, mice skeletal muscle, and liver tissue lysates were prepared using a standard protocol. In brief, the cell and tissue samples were mixed with sample buffer (250 mM tris-hydrochloride (pH 6.8), 0.5 M dithiothreitol (DTT), 10% sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 50% glycerol, and 5% 2-mercaptoethanol) and denatured at 95°C for 5 min. Sample proteins (50 µg) were separated via 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred to nitrocellulose transfer membranes (Whatman, 401396, Dassel, Germany), which were incubated overnight at 4°C with 5% skim milk or 5% bovine serum albumin and a range of antibodies. Anti-AMPK, anti-phospho-(p)-AMPK, anti-AKT, anti-p-AKT, anti-GLUT4 (Bioworld Technology), and anti-β-actin (Bethyl Laboratories) antibodies were the primary antibodies used, and a horseradish peroxidase- (HRP-) conjugated goat anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology) was used as the secondary antibody. The antigen-antibody reaction was detected using a SuperSignal West Femto maximum sensitivity substrate (Thermo Fisher Scientific, Rockford, IL, USA).