Figure 2
From: Stress-induced phospho-ubiquitin formation causes parkin degradation
L-DOPA-induced oxidative stress contributes to parkin loss. (A) Glutathione attenuates L-DOPA’s effect on parkin. Neuronally 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. (B,C) Treatment with carbidopa does not prevent parkin protein loss from L-DOPA treatment. Cells were pretreated with 50 μM carbidopa (carbi) for 2 hours, then co-treated with 200 μM L-DOPA. (B) 16–24 hours after L-DOPA addition, cells were harvested for analysis of intracellular dopamine (DA) levels by HPLC. (C) 24 hours after L-DOPA addition, lysates were harvested for Western immunoblotting. A representative Western blot and quantification of parkin levels are shown. (D) Hydrogen peroxide induces parkin protein loss. Cells were treated with 200 μM hydrogen peroxide for 24 hours before lysates were harvested for Western immunoblotting. (A–D) Error bars show SEM from N = 3 (A), 5 (B), 4 (C) and 3 (D) experiments; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by paired t-test (A–D) with Holm correction for multiple comparisons (A–C). Images of blots have been cropped; uncropped images are shown in Supplementary Fig. 12. Images in A and D show non-adjacent bands from one blot.
