Quantity one image analysis software version 4

Whole proteins were obtained from H9c2 cardiomyocytes with the Protein Extraction Kit (Sigma-Aldrich, St. Louis, MI, USA) by following manufacturer’s protocols, whereas cytosolic and mitochondrial proteins were obtained with the Mitochondria/Cytosol Fractionation Kit (Pierce, Rockford, IL, USA). The protein concentration was quantified by the Lowry method using a Bio-Rad DC protein assay kit II. Equal amounts of proteins were separated by 12% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). After blocking nonspecific binding sites for 1 h with 5% non-fat milk, the membranes were then probed for 2 h at room temperature or at 4 °C overnight with primary antibodies (1:1000 dilution). After washing, the membranes were incubated with appropriate secondary antibodies (1:3000) and detected by an enhanced chemiluminescence kit (Millipore, Boston, MA, USA). The band intensities were analyzed using Quantity One^® image analysis software version 4.62 (Bio-Rad Laboratories, Inc.). To confirm the purity of mitochondrial-cytoplasmic fractionation, the extracts were probed with cytosol-specific anti-tubulin (1:1000) and mitochondria-specific anti-Cox IV (1:1000) antibodies.