Western Blot Analysis of Cardiomyocyte Proteins

After different treatments, proteins from H9c2 cells were obtained with lysis buffer (Thermo Scientific, FL) containing protease inhibitor. The proteins were subjected to electrophoresis and transferred onto nitrocellulose membranes. The membranes were blocked by incubating with 5% dry milk for 1 h, and then incubated with primary antibodies: against β-MHC (1∶1000; Sigma-Aldrich, St. Louis, MO), Akt (1∶500; Cell Signaling Technology, MA), p-Akt (Thr-308, 1∶500; Cell Signaling Technology, MA), eNOS (1∶500; Cell Signaling Technology, MA) or p-eNOS (ser-1177, 1∶500; Cell Signaling Technology, MA), at 4°C overnight. β-actin (1∶4000; Sigma-Aldrich, St. Louis, MO) was used to normalize protein loading. After being washed thoroughly, membranes were incubated with horseradish peroxidase (HRP) conjugated IgG (1∶40000; Jackson ImmunoResearch Labs, INC. PA) for 1 h at RT. Blots were then developed with enhanced chemiluminescence developing solutions and quantified [27] (link).