The destabilization and ubiquitylation of HIF-1α by Fbw7 is dependent on GSK-3β. (A,C) HIF-1α protein levels were measured by Western blot in GSK-3β^+/+ and GSK-3β^−/− cells or GSK-3β^+/+ and GSK-3β^−/− cells transfected with either an expression vector for FlagM2-tagged Fbw7γ or with vectors allowing expression of scrambled (Scr) shRNA or Fbw7 shRNA1 or Fbw7 shRNA2. The HIF-1α protein levels in GSK-3β^+/+ cells were set to 100%. Values are means ± SEM of 3 independent experiments. *Significant difference between shRNA versus scrambled control. (B,D) Representative Western blot analysis. One hundred micrograms of total protein from GSK-3β^+/+ and GSK-3β^−/− cells was analyzed with Abs against HIF-1α, GSK-3α/β, Flag M2, and Golgi membrane (GM). (E) Immunoblot (IB) analysis of anti-V5 immunoprecipitates (IP) and whole-cell lysates (WCE) from HEK 293 cells cotransfected with expression vectors for the V5-tagged full-length HIF-1α mutant HIF-1α P402A/P564A/N803A (PPN), HIF-1α P402A/S551A/T555V/P564A/S589A/ N803A (PPNSTS), and Flag-tagged Fbw7γ (Fγ) or hemagglutinin-tagged ubiquitin.