Fig. 6

The persistence of phosphorylated poly-ubiquitin at the OMM allows for Parkin retention after repolarization of mitochondria. a-e HeLa cells expressing EYFP-Parkin (green) were treated with either a 60 min pulse or continuously with 10 μM CCCP, fixed at the indicated timepoints and stained with DAPI (blue) and antibodies to detect (a) K63 polyubiquitin or (c) K48 polyubiquitin (red) then imaged by confocal microscopy. Scale bars represent 50 μm. For both (a + c), zoomed images of areas represented by the dashed boxes are presented below the ‘merge’ images. The colocalization of EYFP-Parkin with (b) K63 or (d) K48 was assessed using Pearson’s correlation coefficient (PCC) and graphed as the average PCC score. Data is from 3 biological repeats with a minimum of 56 cells per condition. Error is represented as the S.E. Statistical differences in PCC score were appraised by one-way ANOVA and Tukey’s multiple comparisons follow-up test. Statistical significance is indicated as follows: *, p < 0.05; **, p < 0.01. E, Western blot analysis for phospho-polyubiquitin levels in EYFP-Parkin expressing HeLa cells treated with either a 60 min pulse or continuously with 10 μM CCCP