Anti phospho camkii

Muscles were homogenized by hand in a Dounce homogenizer in reducing sample buffer (80 mM Tris, pH 6.8, 10% β-Mercaptoethanol, 2% SDS and 10% glycerol) with phosphatase inhibitor (Thermo scientific) and protease inhibitor cocktail (Sigma). An equal amount of total protein was loaded on SDS-PAGE gels followed by transfer to nitrocellulose membrane. Ponceau red S was used to verify the transfer onto the nitrocellulose. The following primary antibodies were used for western blotting: anti- β-CaMKII (1:500, Life Technologies), anti- phospho-CaMKII (1:750, Thermo Scientific), anti-AMPK (1:1000), anti-phospho (T172) AMPK (1:1000), anti-Akt (1:1000), anti-phospho Akt (1:500)(Cell Signaling). Secondary antibodies conjugated with HRP were from Sigma-Aldrich (used 1:10000). Specific signals were developed using ChemiGlow chemiluminescent substrate for HRP (Protein Simple). Images of the blots were acquired using FluorChem FC2 Imager (Alpha Innotech). Specific signals were developed using ChemiGlow chemiluminescent substrate for HRP (Protein Simple). Images of the blots were acquired using FluorChem FC2 Imager (Alpha Innotech). Quantitative analysis was performed using ImageJ software.