Figure 8

Phospho-Ub-dependent parkin loss does not require its activity in cis. (A–I) Differentiated PC12 cells were transduced with lentiviral vectors carrying wild-type or C431S parkin. Three to five days after transduction, cells were treated with 10 μM CCCP for 6 hours (A–C) or 12 hours (C), 200 μM L-DOPA for 24 hours (D–F), or 200 μM hydrogen peroxide for 24 hours (G–I) before being harvested for Western immunoblotting. Representative Western immunoblots and quantifications of phospho-poly-Ub (B,E,H) and parkin (C,F,I) levels are shown. Error bars show SEM from N = 5 (B), 6 (C, left), 4 (C, right), 3 (E), 6–8 (F), 5 (H,I) independent experiments; *p ≤ 0.05, **p ≤ 0.01, ****p ≤ 0.0001 by paired t-test with Holm correction for multiple comparisons (B,E,H) or relative to WT by one-way ANOVA of drug-treated mutants with Holm-Sidak’s multiple comparisons test (C,F,I). The leftmost half of (G) also appears in Fig. 4D. Images of blots have been cropped; uncropped images are shown in Supplementary Fig. 12.