Total creb

The heart tissue was homogenated in extraction buffer (20 mmol/L Tris HCl, 1 mmol/L Na 3 VO 4 , 5 mmol/L NaF). The heart protein was collected and subjected to 10% or 15% SDS-polyacrylamide gel electrophoresis then transferred to polyvinylidene difluoride membranes, which were blocked for 2 h with 5% non-fat milk in Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100. The membranes were probed overnight at 4 C with the appropriate primary antibody as follows: total-p38, phospho-p38, and phospho-HSP27 (Cell Signaling Technology, Danvers, MA), total-p53, phospho-p53, total-CREB, phospho-CREB, Bax, Bcl2, Cytochrome c, caspase-3 (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). After washing and exposure to horseradish peroxidaseconjugated secondary antibody for 1 h at room temperature, antibody-antigen complexes were visualized by enhanced chemiluminescence assay. Bands corresponding to the protein of interest appeared as dark regions on the developed film. The film images of the Western blots were scanned and were analyzed using Image J (NIH image, National Institutes of Health, Bethesda, MD) analysis software (Tanno et al., 2003) . For quantitation of the proteins of interest, phosphorylated proteins were normalized to total protein expression.