P akt2

The total protein from the tissue and H9C2 cardiomyocytes was extracted using a cell lysis solution (Beyotime Institute of Biotechnology) and phosphatase inhibitor (Meilun Bio, Dalian, USA) and quantified using the Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime). An equal amount of protein (40 mg/well) was separated by 10% and 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes for 1.5 hours at 100 V. The membrane was then blocked with 5% BSA at room temperature for 1.5 hours. GAPDH, akt2, p-akt2, cleaved caspase3, bax, and bcl-2 primary antibodies were incubated with the membranes at 4°C overnight (Akt2; 1 : 200; Santa Cruz Biotechnology, Santa Cruz, CA, USA; p-akt2; 1 : 500; Abcam, Cambridge, MA, USA; cleaved caspase3; 1 : 500; Cell Signaling Technology, Danvers, MA, USA; bax and bcl-2; 1 : 500; Proteintech; GAPDH; 1 : 10000; Abways). Subsequently, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Proteintech) at room temperature for 2 hours. The blotting was visualized using enhanced chemiluminescence (ECL detection kit, KeyGEN Biotech, Jiangsu, China) on the c300 Chemiluminescent Western Blot Imaging System (Azure Biosystems, Dublin, GA, USA).