Western blot analysis of AMPK and p-AMPK

Total liver protein was isolated by phosphorylated protein extraction kits including phosphatase inhibitors and a protease inhibitor. The protein content on the lysates was estimated by the coomassie brilliant blue method. Proteins (80 µg) were separated with 10% SDS-PAGE and then transferred to a PVDF membrane (2 h, 200 mA). The membranes were blocked in 5% BSA for 1 h at room temperature. Then, the membranes were incubated overnight with a primary antibody against AMPK (1:1,000, Cell Signaling Technology, Beverly, MA) and Thr¹⁷²-phosphorylated AMPK (1:1,000, Cell Signaling Technology) in blocking buffer. After washing in Tris-buffered saline/Tween 20 under gentle agitation, the membranes were incubated for 1 h with a horseradish peroxidase-labelled anti-rat IgG (1:10,000). After further washing, blots were treated with enhanced chemiluminescence detection reagents. Blot intensities were measured using Image J software (NIH).