Western blot analysis
A total of 200 mg of liver and skeletal muscles were lysed with 1 ml RIPA buffer and followed by centrifugation at 13,000 r/min for 10 min. The supernatants were collected and protein concentrations were measured using 660 nm Protein Assay Reagent kit (ThermoFish scientific, China). A 3 μg/μL of protein extract was supplied with lysis buffer and denatured by boiling at 95 °C for 10 min. The denatured proteins were resolved by 12% and 10% SDS-PAGE and transferred to nitrocellulose membrane. The membranes were blocked with 5% skim milk at room temperature for 2 h, incubated with primary antibodies overnight at 4 °C, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h. The protein bands were visualized and quantitated using the Image Jet software and with β-actin as an internal standard.