West pico plus chemiluminescence substrate

Total protein levels in the total, nuclear, and cytoplasmic samples were measured using the Pierce™ BCA Protein Assay Kit (Cat # 23225, ThermoFisher, USA). Proteins were diluted in the loading buffer and then equal volumes of each sample were separated by the SDS-PAGE. The proteins were then transferred to nitrocellulose membranes, blocked with 5 % skimmed milk, and incubated with primary antibodies against total and phospho-AMPK (Cat # 2532 and Cat # 2531; Cell Signaling Technology, USA; 62 kDa, 1:1000 & 1:500, respectively) or β-actin (# 3700, 45 kD, 1:1000). The membranes were then incubated with the corresponding secondary antibodies and incubated with West pico PLUS chemiluminescence substrate (Cat # 34580, ThermoFisher, USA) for 5 min the developed bands were scanned and analyzed using the C-Di Git blot scanner. Washing three times each of 10 min with TBST buffer was conducted between steps. All antibodies, as well as the skimmed milk, were diluted in the TBST buffer. Incubations with the primary or secondary antibodies were performed at room temperature for 2 h and with continuous shaking. The expression of all target proteins was normalized against β-actin.