Figure 5.

Parkin and PINK1 are required for ubiquitination of MFN-1 and MFN-2. (A) Control or Parkin KD cells were treated with vehicle or CCCP for 2 h, cell lysates prepared and MFN-1 or (B) MFN-2 ubiquitination assessed by western blot using antibodies against MFN-1 and MFN-2. (C) SH-SY5Y cells were untransfected (UT), transfected with PINK1 siRNA #1 or PINK1 siRNA #2 or transfected with control siRNA for 72 h. Untransfected cells and siRNA transfected cells were then treated with 10 µm CCCP for 2 h, cell lysates prepared and separated by western blot. Blots were probed with an antibody against MFN-1 or (D) MFN-2. Blots were re-probed with an antibody against SDHA for mitochondrial content. Blots are representative of at least three separate experiments. Ubiquitinated species are denoted by arrowheads.