Figure 3.

VDU2 deubiquitinates HIF-1α. (A) Deubiquitination of HIF-1α by VDU2 in vitro. The purified polyubiquitinated HIF-1α was incubated with the purified recombinant VDU1 or VDU2 in a deubiquitination buffer for the durations indicated at 30°C followed by western blot with anti-HIF-1α antibody. (B) Regulation of HIF-1α ubiquitination levels in vivo. Western blot analysis of immunoprecipitates with anti-HIF-1α antibody from HEK293 cells transfected with HIF-1α and HA–ubiquitin (lanes 1–5), pVHL (lanes 3–5), VDU1 (lane 4), VDU2 (lane 5), and treated with MG132 (lanes 2–5). The blot was detected with anti-HA antibody.