Western blot detection kit

Either the RIPA or 1% NP-40 lysis buffers containing protease inhibitor cocktail tablets (Roche) were used to extract proteins from murine muscle tissue and C2C12 cells, respectively. Moreover, the buffers were centrifuged for 20 min at 12,000 rpm at 4 °C to eliminate the pellet. After quantifying the protein by the Bradford assay method, the protein was separated by size using sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transferring the protein to the polyvinylidene fluoride membrane by the transfer system (Bio-Rad, Hercules, CA, USA), the blocking buffer (0.5% skim milk, 1× TBS buffer) containing 5% skim milk was processed for 1 h. The membranes were washed thrice with 1× Tris-buffered saline (TBS) for 10 min and incubated with primary antibodies for proteins at 4 °C overnight. The membranes were then washed thrice with 1× TBS buffer for 10 min. The protein bands were measured with the Western blot detection kit (Bio-Rad), and the chemiluminescence imaging system (ImageQuant LAS 4000; GE Healthcare, Munich, Germany) equipment was used to observe the expression level. Moreover, the membranes were washed thrice with 1× TBS buffer for 10 min. The protein bands were detected using the Western blot detection kit (Bio-Rad) and the chemiluminescence imaging system (GE Healthcare).

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).