Western Blot

Heart tissues and cardiomyocytes were lysed in RIPA buffer. Proteins were isolated as previously described (Liu et al., 2015). Briefly, the left ventricles were polished and the cardiomyocytes were lysed in RIPA buffer, and then, the protein concentrations were measured using the BCA Protein Assay Kit (Thermo, 23227) and an ELISA Reader (Synergy HT, Bio-Tek). The cell lysates (50 mg) were loaded into each lane and subjected to SDS-PAGE, and the proteins were then transferred onto Immobilon-FL membranes (Millipore, IPFL00010). The membranes were incubated overnight at 4°C with primary antibodies against one of the following proteins: phosphorylated (p-) and total (T) PI3K, Akt1, AMPKα, AMPKα, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase beta-1 (70S6K), S6, extracellular regulated protein kinases (ERK)1/2, NFAT2 and GAPDH (all of the antibodies were purchased form Cell Signaling Technology and diluted at 1:1000). The blots were scanned using a two-color infrared imaging system (Odyssey, LI-COR). Specific protein expression levels were normalized to the GAPDH protein for the total cell lysates and cytosolic proteins.