Figure 5. USP9X down-regulates pVHL through Smurf1.

A. HEK293T cells were transfected with constructs as indicated and harvested after 36 hours. Harvested cells were immunoblotted with indicated antibodies. B. CAKI cell lysates were subject to immunoprecipitation with anti-USP9X or Smurf1 antibodies. Immunoprecipitates were then blotted. C. HEK293T cells transfected with the indicated constructs were treated with MG132 (10 μM) for 8 h before harvest. Cell lysate was subjected to pulldown with Ni-NTA beads and western blot with anti-Flag antibody to evaluate for ubiquitinylated pVHL.