Chemiluminescent kits

Cells were lysed in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail). Equivalent protein quantities were subjected to SDS-PAGE, and transferred to PVDF membranes (Millipore). Membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with the indicated primary antibodies overnight at 4 °C, followed by the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. After washing with TBST three times, the blotted membranes were visualized with chemiluminescent kits (Millipore).

The total cardiomyocyte cellular protein was loaded onto an 8–12% sodium dodecyl sulfate-polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes (Millipore, USA). The membranes were sealed and incubated with particular primary antibodies targeting phosphorylated phosphatidylinositol 3-kinase (p-PI3K) (rabbit polyclonal, ab70912; Abcam), phosphatidylinositol 3-kinase (PI3K) (rabbit polyclonal, ab191606; Abcam), phosphorylated protein kinase B (p-AKt) (rabbit polyclonal, ab38449; Abcam), protein kinase B (AKt) (rabbit polyclonal, ab64148; Abcam), phosphorylated mammalian target of rapamycin (p-mTOR) (rabbit polyclonal, ab226957; Abcam), mammalian target of rapamycin (mTOR) (rabbit polyclonal, ab32028; Abcam), B-cell lymphoma 2 (Bcl-2)(rabbit polyclonal, ab32124; Abcam), Bcl-2 associated X protein (Bax) (rabbit polyclonal, ab32503; Abcam), caspase3 (rabbit polyclonal, ab13847; Abcam), cleaved-caspase3 (rabbit polyclonal, ab2302; Abcam), and β-actin (rabbit polyclonal, ab8227; Abcam) overnight at 4°C. The protein expression of the membranes was visualized using chemiluminescent kits (Millipore, USA) via ultrasensitive chemiluminescence imager (GE Amersham Imager 600, USA). The gray-scale values of the target bands were analyzed by Image J.