Figure 4.

Parkin ubiquitinates p21 in vivo and in vitro. A, Myc-parkin and Flag-p21 were cotransfected into 239T cells. At 36 hours after transfection, cell lysates were harvested and was immunoprecipitated with anti-Myc or control IgG. Western blotting was performed (left panel). Parkin interacts with endogenous p21. Cell extracts of neural stem cells were immunoprecipitated with anti-Myc or control IgG. The immunoprecipitated complex was detected by Western blotting with anti-p21 (right panel). B, Parkin and ubiquitin proteasome system regulate p21 levels. Steady-state p21 levels were significantly decreased by coexpressing increasing amounts of parkin in HEK293 cells (upper panel). In the presence of cycloheximide, coexpression of parkin with p21 led to an accelerated decrease of p21 steady state levels compared with p21-alone transfected control (lower panel). For RT-PCR, total RNA were obtained from the transfected cells. The expression of p21 mRNA was determined by RT-PCR. GAPDH was internal control. C, 293T cells were cotransfected with constructs as indicated. At 48 hours after transfection, cells were treated with MG132 (10 μM) for 6hr, then harvested and immunoprecipitated with anti-p21. Ubiquitinated p21 was visualized by Western blot analysis using anti-ubiquitin (left panel). Parkin and Ubc5 mediate p21 ubiquitination. The ubiquitination reaction buffer, E1, UbcH5a, and parkin were incubated with p21 and polyubiquitinated p21 was detected by Western blotting (right panel). D, Endogeneous ubiquitination of p21 in non-tg or PARK2 KO mice. Neural stem cells isolated from non-tg or PARK2 KO mice were differentiated for 5 days, and then treated with MG132 (10 μM) for 6hr, then harvested and immunoprecipitated with anti-p21. Ubiquitinated p21 was visualized by Western blot analysis using anti-ubiquitin. Each band is representative for three experiments.