Mitochondrial and Cytosolic Fractionation

Mitochondrial and cytosolic fractions were generated by homogenizing freshly excised hearts in homogenization buffer (20 mM HEPES, 140 mM KCl, 10 mM EDTA, 5 mM MgCl₂, pH 7.4) with a Dounce tissue homogenizer, centrifuging the homogenate at 800 × g for 10 minutes and centrifuging the resulting supernatant at 8,000 × g for 10 minutes [77 (link)]. The supernatant is the cytosolic fraction. The pellet was washed by centrifugation at 10,000 × g and represents the mitochondrial fraction. Whole-cell extracts and mitochondrial membranes were prepared as described previously. Samples were loaded on SDS-PAGE, transferred to nitrocellulose or PVDF membranes, and incubated with specific antibodies [78 (link)]. Bands were visualized using horseradish peroxidase–conjugated secondary antibodies and the ECL detection system (GE Healthcare, Piscataway, NJ) or fluorophore-conjugated secondary antibodies and the Odyssey fluorescence detection system (Li-Cor Biosciences, Alpharetta, GA).