Figure 4.

MG132 induces the formation of insoluble, high-molecular-weight Parkin species. COS-7 cells were transfected with FLAG-Parkin (left), FLAG-HHARI (right), or mock-transfected (middle) and grown in the presence or absence of MG132 for 16 or 28 h post-transfection. Cell lysates were prepared in buffer as indicated, and 10 μg of each soluble (S) and insoluble (I) fractions was separated by SDS-PAGE and analyzed by Western blotting. Blots were probed with mouse anti-FLAG (top), affinity-purified rabbit anti-UbcH7 peptide (1:5000) (middle), and anti-β-actin (bottom) antibodies. Arrows indicate the presence of each respective FLAG construct.