Total ampk

For Western blot analysis, cells were homogenized in a cell lysis buffer (200 μL) with 30 min interval ice incubation, and then centrifuged at 14,000 rpm, at 4 °C for 20 min. Protein concentrations were determined using the Bradford Protein Assay Kit. The proteins were then resolved by SDS-PAGE (10%) (Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis), transferred to a nitro cellulose membrane using the semi-dry transfer system (Biorad, Hercules, CA, USA) and treated with a blocking solution (0.5% skim milk, 1 × Phosphate-buffered saline with Tween 20 (PBST) buffer) for 1 h. The membrane was then washed three times for 10 min in PBST buffer, incubated overnight (4 °C) with primary antibodies against total-AMPK, p-AMPK (Thr172) (Santa Cruz Biotechnology, Dallas, TX, USA) LDH (Santa Cruz, CA, USA), SDHA (Santa Cruz, CA, USA) and PGC-1α (Santa Cruz, CA, USA), and then washed again with the aforementioned procedure. Following this, the membranes were probed with secondary antibodies and washed with PBST buffer. After treatment with the buffer, protein band intensity was detected using the enhanced chemiluminescence Chemi-Doc, XRS system (BIORAD, Hercules, CA, USA) and compared with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and total-AMPK after activation using a Western blot detection kit (Biorad, USA).