Cells were lysed with lysis buffer (50 mM Tris HCl, pH 7.5, 0.1 M NaCl, 1 mM EDTA, 1% Triton X-100, 10 mg/ml each of aprotinin and leupeptin, and 1 mM PMSF). In some cases, mitochondrial fractions were lysed with the lysis buffer. A portion of cells (20-30 mg) or mitochondrial lysates (10 mg) were separated on 12% SDS-PAGE, transferred onto nitrocellulose membranes (Schleicher and Schuell), and subjected to western blot analysis by using designated primary antibodies. Horseradish peroxidaseconjugated secondary antibodies (Cell Signaling Technology, Beverly, MA) followed by ECL (Amersham Biosciences Inc., Piscataway, NJ) were used for detection.