Figure 4

Association with oxidative stress-induced phospho-Ub leads to parkin loss. (A,B) Differentiated PC12 cells were transduced with lentiviral vectors carrying the indicated parkin mutants. Three to five days after transduction, cells were treated with 200 μM L-DOPA for 24 hours before harvest for Western immunoblotting and assessment of parkin protein levels. Representative immunoblots (A) and quantifications of parkin levels (B) are shown. The level of each parkin mutant after L-DOPA treatment was normalized to the level of the same mutant after control treatment, which was set to 1 in each case. The latter is represented by the leftmost gray bar in the quantifications (B). (C) Glutathione almost completely abrogates the L-DOPA-induced phospho-poly-Ub signal. Differentiated PC12 cells were co-treated with 200 μM L-DOPA and 200 μM glutathione (GSH) for 24 hours before lysates were harvested for Western immunoblotting. A representative immunoblot and quantification of phospho-poly-Ub levels are shown. (D,E) Differentiated PC12 cells were transduced with lentiviral vectors as in (A) and treated with 200 μM hydrogen peroxide for 24 hours before harvest for Western immunoblotting. (D) Hydrogen peroxide induces phospho-poly-Ub formation and decreases wildtype overexpressed parkin. (E) Effect of hydrogen peroxide on overexpressed parkin mutants. Representative immunoblot and quantifications of parkin levels as in (B) are shown. (B,C,E). Error bars show SEM from N = 3–8 (B), 5 (C), and 5 (E) independent experiments; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by paired t-test with Holm correction for multiple comparisons (C) or relative to WT by one-way ANOVA of stressor-treated mutants with Holm-Sidak’s multiple comparisons test (B,E). Images of blots have been cropped; uncropped images are shown in Supplementary Fig. 12. For A, the S65A and K151E images came from a different blot than the WT/H302A image. S65A and K151E are from the same blot at different exposures.