Complete protease inhibitor cocktail minus edta

Cytosol fractions were isolated from HeLa cells transfected with Parkin, ParkinΔUBL, ParkinS65A, or ParkinS65E. Cells were homogenized in Solution B (20 mM Hepes-KOH, pH 7.6, 220 mM mannitol, 70 mM sucrose, and 10 mM KOAc) supplemented with complete protease inhibitor cocktail minus EDTA (Roche). Homogenates were centrifuged at 800 g for 10 min at 4°C and the following supernatant was then centrifuged at 100,000 g for 30 min at 4°C to obtain the final cytosolic fraction. Mitochondria were isolated from HeLa cells expressing PINK1-V5-His in the absence of Parkin that were untreated or treated with 10 µM CCCP for 3 h. Cells were homogenized in Solution B and centrifuged at 800 g for 10 min at 4°C and the following supernatant was then centrifuged at 10,000 g for 20 min at 4°C to obtain the mitochondrial fraction.
Cytosolic extracts from HeLa cells were supplemented with 1 mM DTT and ATP-regenerating buffer from a 10× stock solution (20 mM Hepes-KOH, pH 7.6, 10 mM ATP, 300 mM phosphocreatine, 10 mM MgCl₂, 10% glycerol, and 1.5 mg/ml creatine phosphokinase). Mitochondria (35 µg) were resuspended in 10 µl of energized cytosol and incubated at 30°C for 90 min. The reactions were then stopped with 10 µl of 2× LDS sample buffer (Invitrogen) supplemented with 200 mM DTT.