Frozen mouse cardiac tissue was homogenized with lysis buffer containing 50 mmol/L Tris-HCl (pH7.5), 5 mmol/L EDTA, 10 mmol/L EGTA, 1X cock tail protease inhibitor, 1X alkaline phosphatase inhibitor and 1X acid phophatase inhibitor, 50 ug/ml phenylmenthysulfonyl fluoride and 1.23 mg/ml Chaps. Extracts were centrifuged at 12,000 rpm at 4°C for 15 minutes. 10 ug of the sample proteins was mixed with loading buffer (40 mmol/L Tris-HCl, pH 6.8, 1% SDS, 50 mmol/L DTT, 7.5% glycerol and 0.003% bromophenol blue and heated at 95°C for 5 minutes, and subjected to electrophoresis on a gradient gel (4% to 12%) at 120V. After electrophoresis, the protein was transferred to a PVDF membrane in a transfer buffer. The PVDF membrane was rinsed briefly in TBS buffer containing 50 mM Tris, 137 mM NaCl, pH 7.5 and blocked in buffer (5% milk with 0.5% BSA in TBST buffer (TBS buffer containing 0.1% tween 20) at room temperature for 1 hour. The membrane was then incubated with rabbit anti 4-hydroxy-2-noneal (4HNE) antibody at 1/3000 dilution at 4°C over night, followed by washing three times. The secondary antibody was incubated with the membrane for another one hour at room temperature. Finally the antigen-antibody complexes were visualized with use of an enhanced chemiluminescence kit. Anti-GAPDH (Abcam) was used for normalizing.