Rabbit anti ampk

The total proteins were extracted using lysis buffer. Equal amounts of the protein were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes. The membranes were blocked for 1 h at room temperature using 5% nonfat milk and were incubated overnight at 4℃ in the appropriate primary antibody, which included rabbit anti-p-ACC (abcam, 1:250), rabbit anti-ACC (abcam, 1:250), rabbit anti-p-AMPK (abcam, 1:250), rabbit anti-AMPK (abcam, 1:250) and rabbit anti-GAPDH (abcam, 1:250). Then, the membranes were washed 3 times for 10 min each with Tris-buffered saline containing Tween and were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Following additional washes, the immunoreactive proteins on the membrane were visualized with enhanced chemiluminescence plus reagents (Millipore, Plano, TX, USA). The intensities of the immunoreactive proteins were measured via image J and were normalized to GAPDH. All the experiments were repeated at least 3 times.