Figure 2.

The anti-apoptotic effects of parkin are intrinsic to the mitochondria and can be observed in a cell-free system. (A) Human parkin was stably expressed in two independent monoclonal MES cell lines. Whole cell lysates were probed for parkin and actin expression by western blot. (B) Mitochondria were isolated from MES, MES myc-Parkin and MES HA parkin cells, protein normalized and incubated with DMSO, or 10 µm of the purified BH3 domain peptides of Bid and Bim at various concentrations. NP-40 (1%) was added to aliquots of each mitochondrial preparation to measure total cytochrome c from each sample. Released cytochrome c was analyzed by western blot. (C) Caspase 3/7 activity was measured from MES and MES myc-Parkin cells 18 h after the treatment with vehicle or 1 µm C2 ceramide (mean ± SEM, n = 20). Data were pooled across three independent experiments, # denotes statistically significant from control (P < 0.00001). (D) MES, MES myc-Parkin and MES HA-Parkin cells were treated with vehicle, 1 µm C2 ceramide or 300 nm staurosporine for 6 h and caspase 3/7 activity was measured (mean ± SEM, n = 4), *denotes statistically significant from control (P < 0.001).