Figure 6

Phospho-poly-Ub is present in both cytosolic and mitochondrial fractions and leads to mitochondrial parkin translocation. (A–F) PC12 cells were treated with 200 μM L-DOPA for 14–17 hours (A,C,E) or 10 μM CCCP for 6 hours (B,D,F) before being harvested for sub-cellular fractionation. 15% of the resulting cytosolic fractions and 100% of the mitochondria-enriched fractions were analyzed by Western immunoblotting for the indicated proteins. (A,B) Representative Western blots showing the distribution of phospho-ubiquitin, parkin, GAPDH, UQCRC1, and Tom20 between cytosolic and mitochondrial fractions. (A–D) Phospho-poly-ubiquitin is present both in the cytosol and on mitochondria after L-DOPA (A,C) and CCCP (B,D) treatment. The phospho-poly-ubiquitin signal in the cytosolic and mitochondrial fractions was normalized to the percentage of the fraction that was loaded to estimate the total level of phospho-poly-ubiquitin in each fraction. The same quantification was carried out for mitochondrial (Tom20, UQCRC1) and cytosolic (parkin, GAPDH) proteins from untreated cells to confirm that fractionation was successful. (A,B,E,F) Parkin protein translocates to mitochondria after L-DOPA (A,E) and CCCP (B,F) treatment. Parkin levels in the mitochondrial fraction with drug or control treatment were normalized to levels of UQCRC1 and plotted in (E,F). (C–F) Error bars show SEM from N = 4 (C,D), 7 (E), and 4 (F) independent experiments; for F, one outlier was identified using the ROUT method with Q = 0.5% and omitted. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 by paired t-test. Images of blots have been cropped; uncropped images are shown in Supplementary Fig. 12.