Whole Cell Lysate Preparation and Western Blotting

Detailed experimental procedures have been described previously [4 (link)]. Briefly, whole cell lysates were prepared by resuspending cell pellets in cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin). Protein concentration was determined using Pierce BCA Protein Assay Kit (Pierce, Rockford, IL). Protein extract was loaded onto an SDS-polyacrylamide gel, separated by electrophoresis and then transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was then incubated with primary antibodies against AMPKα, phospho-AMPKα (Thr172) and phospho-ACC (Ser79) (Cell signaling, Technology Inc, Beverly, MA) and γ-tubulin (Sigma-Aldrich, St Louis, MO) overnight at 4°C. After washing with TBS-T, the membrane was incubated with rabbit IgG secondary antibodies, and the signals were visualized using the ECL western blotting system (Millipore, Billerica, MA).