Western blot analysis.

Heart tissue samples were prepared, supernatants were collected, and gel electrophoresis was performed as previously described (1). Proteins from gels were transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and blocked with 5% milk. Membranes were incubated with the following desired primary antibodies: matrix metalloproteinase (MMP)-9 (1:1,000, catalog no. sc-6840, Santa Cruz Biotechnology) (7, 14, 31), p-AKT (1:1,000, catalog no. 4085L, Cell Signaling) (17, 18, 50), AKT (1:1,000, catalog no. 4685S, Cell Signaling) (8, 9, 22), p-PTEN (1:1,000, catalog no. 9554S, Cell Signaling) (13, 34, 59), PTEN (1:1,000, catalog no. 9559L, Cell Signaling) (6, 15, 25), cleaved caspase-3 (1:1,000, catalog no. 9661L, Cell Signaling) (24, 53, 55), and β-actin (as a loading control, 1:1,000, catalog no. 4967L, Cell Signaling) (12, 32, 54) for 3 h at room temperature or overnight at 4°C. Membranes were then washed and incubated with the appropriate secondary antibody for 1 h at room temperature. Finally, membranes were washed again, incubated in chemiluminescent ECL substrate (Fisher), and exposed using developing film. Band intensities were measured using ImageJ software and are presented as arbitrary units.