Parkin

Cells were seeded in growth medium and left untreated or treated with 12.5 μM CCCP, after 24 h the medium was washed out and replaced with fresh medium containing 12.5 μM CCCP and incubated 24 h more (48 h total). Medium was then aspirated from each dish and cells washed twice with cold PBS. Cells were lysed in 60 μL RIPA buffer with protease inhibitors. Unlysed cells and debris were pelleted for 15 min at 16,000 xg at 4˚C. Samples were separated using SDS-PAGE and transferred to nitrocellulose membranes. Transfer was performed using the iBlot system (Invitrogen). Membranes were treated with Li-COR Odyssey blocking buffer for 1 h at room temperature, then incubated with primary antibody in a 1:1 solution of PBS-T and Li-COR odyssey blocking buffer overnight at 4˚C. Following three 5 min washes in PBS-T, the membrane was incubated with secondary antibodies (1:3000) in a 1:1 solution of PBS-T and Li-COR Odyssey blocking buffer for 1 h at room temperature. Following three 5 min washes in PBS-T, membranes were scanned using the Li-COR Odyssey Imaging System. Antibodies for cytochrome c (BD Biosciences), tom20 (Santa Cruz) and parkin (Sigma-Aldrich) were detected using a goat anti-rabbit or goat anti-mouse IgG antibody conjugated to an IRdye at 800CW and 680CW conjugated, respectively (Li-COR Biosciences).