AMPK activation by Western blot analyses of liver and skeletal muscle proteins

Approximately 150 mg of frozen liver and muscle tissues were homogenized in tubes containing RIPA buffer with protease and phosphatase inhibitors plus ceramic beads (1.4 mm Ceramic, Bulk Beads, Fisher Scientific, Pittsburgh, PA, USA) using a Precellys 24 (PEQLAB Biotechnology GmbH, Germany). Liver tissue was homogenized for 1 × 30 sec at 6,500 rpm whereas muscle was homogenized for 3 × 30 sec at 6,500 rpm with 20 sec intervals. The homogenates were centrifuged at 15,700 × g for 10 minutes and aliquots (50 µg of protein) of the supernatant (lysate) were separated by SDS-PAGE using 4–15% MP TGX Gels (Bio-Rad # 4561083) and blotted onto nitrocellulose membranes. Membranes were blocked with 5% nonfat milk for 2 h and incubated with antibodies against pAMPK (Phospho-AMPKα (Thr172) Antibody, cat. no. #2531 Cell Signaling Technology, Danvers, MA, USA), AMPK (AMPKα Antibody, cat. no. #2532 Cell Signaling Technology, Danvers, MA, USA), or β-actin (Santa Cruz Biotechnology) for 1 h or overnight. After a second reaction with secondary antibodies (horseradish peroxidase-conjugated IgG goat antirabbit or goat antimouse antibodies), the protein bands on the nitrocellulose membranes were visualized by enhanced chemiluminescence substrate on a Biorad ChemiDocTM MP Imaging System (BioRad Laboratories Inc, Hercules, CA, USA). Band intensities were quantified by densitometry using Image J software.