Lack of Fbw7 induces HIF-1α protein levels. (A,C) HCT116 Fbw7^+/+ and HCT116 Fbw7^−/− cells were cultured under normoxia (16% O₂) for 24 hours and then further cultured for 4 hours under normoxic or hypoxic (5% O₂) conditions. The HIF-1α protein levels measured by Western blot under normoxia (16% O₂) were set to 1. Values are means ± SEM of 3 independent experiments. *Significant difference between 16% O₂ versus 5% O₂; **significant difference between 5% O₂ Fbw7^+/+ or 5% O₂ Fbw7^−/− versus 5% O₂ + Fbw7. (B,D) Representative Western blot analysis. One hundred micrograms of total protein from HCT116 Fbw7^+/+ and HCT116 Fbw7^−/− cells was analyzed with Abs against HIF-1α, c-Myc, Flag M2, and Golgi membrane (GM). (E) HepG2 cells were transfected with an expression vector for either FlagM2-tagged Fbw7γ (γ), Fbw7WD (WD), or control vector, and PAI-1 levels were measured by Western blot. The PAI-1 protein level in the control was set to 100%. Values are means ± SEM of 3 independent experiments. *Significant difference in the control vector versus Fbw7γ and Fbw7WD. (F) Representative Western blot. One hundred micrograms of protein from the medium and 100 μg of total protein extract from HepG2 cells transfected as in panel E were analyzed with the hPAI-1, FlagM2, and albumin Abs, respectively.