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.