Western Blot Analysis of Cellular Signaling

Western blot analysis was performed using the method described previously^{21 (link)}. Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a microporous polyvinylidene fluoride membrane (Millipore Corporation, MA, USA) for western blotting^{21 (link)}. The membrane was incubated at 4 °C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPK, AMPK, p-ACC, ACC (all at 1:1000), and β-actin (1:2000)^{21 (link)}. After incubation, anti-mouse (1:2000 for β-actin) or anti-rabbit IgG horseradish peroxidase conjugate (1:2000 for p-Akt, Akt, p-AMPK, AMPK, p-ACC, and ACC) was added, and the membrane was incubated for 30 min at RT. The antigenic proteins on the membrane were visualized via chemiluminescence using a Lumi-Light^PLUS Western Blotting Kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan)^{21 (link)}.