Western Blot Analysis of Cardiac Proteins

Equal protein amounts from isolated cardiomyocytes, rat heart, and isolated mitochondria were resolved by 8-12% SDS-PAGE and transferred to polyvinylidene fluoride membrane for immunoblot analysis, as previously described [26 (link)]. Membranes were blocked with 5% nonfat milk in Tris-Buffered Saline- (TBS-) Tween and were incubated with primary antibodies overnight at 4°C at the following dilutions: AMPK, phosphorylated AMPK (Thr172), STAT3, phosphorylated STAT3 at Tyr705 (p-STAT3 Tyr705), Bax, Bcl2, caspase-3, and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA) 1 : 1000; phosphorylated STAT3 at Ser727 (p-STAT3 Ser727) (Cell Signaling Technology, Beverly, MA) 1 : 500. After washing with TBS-Tween, immunoreactive bands were visualized by an enzymatic chemiluminescence method and quantified with Quantity One image software.