FIGURE 8.

hFis1 is ubiquitinated by parkin and degraded by proteasome. Total cell lysates from HEK 293 cells transfected with indicated plasmids for ∼48 h in the presence or absence of 10 μm of MG132 for ∼24 h were immunoblotted (IB) with anti-HA, anti-Myc, and hFis1 antibodies (Input panel). Tubulin and Hsp60 were used as the loading control. Immunoprecipitation (IP) was performed with a mouse monoclonal anti-HA antibody. When immunoblotted with a polyclonal anti-hFis1 antibody, a single band of hFis1, shifted from ∼16 to ∼24 kDa was detected in cells transfected with ubiquitin and dramatically increased when parkin and MG132 were also present. * denotes nonspecific band. Experiments were repeated twice.