Figure 5. Mitochondrial ubiquitination by Parkin is essential for VCP recruitment.
A. Parkin-dependent mitochondrial ubiquitination. HeLa cells were transfected with EGFP-Parkin wt or Parkin T240R, treated with 10 µM CCCP for 10 h and immunostained for cytochrome C (red, mitochondria) and ubiquitin (blue). Scale bars equal 10 µm. See also Supplemental Figure 6. B. Micrographs of HeLa cells expressing VCP-mCherry and either EGFP-Parkin wt or Parkin T240R. The images are taken during the mitochondrial aggregation stage after CCCP treatment (see Supplemental Figure 6 for the full time course). Scale bars equal 10 µm. C. Quantification of VCP recruitment to mitochondria following CCCP treatment in HeLa cells expressing either Parkin wt or Parkin T240R. Error bars indicate standard error from 3 replicates. 30 cells were counted for each replicate.