Figure 6.

Knockdown of BRAF and translation of HIF-1α and HIF-2α. A, HT29 pSicoR cells were cultured in normoxia and treated with the proteasome inhibitor MG132 for the indicated times. Protein levels of HIF-1α HIF-2α and β-actin were analyzed by western blotting (upper panel). Band densities were quantified and HIF-1α and HIF-2α protein levels were normalized to β-actin at each time point. Protein synthesis of HIFs was measured by its accumulation during MG132 treatment and data represent the mean ± SD for 2 independent experiments (lower panels). *P<0.05. B, Inhibition of MAPK and translation of HIF-1α and HIF-2α. HT29 cells were treated with DMSO or PD98059 in normoxia, and then treated with MG132 for the indicated times. Protein levels of HIF-1α and HIF-2α were analyzed by western blotting (upper panel). Protein synthesis of HIFs was measured as indicated above and data represent the mean ± SD for 2 independent experiments (lower panels). *P<0.05. C, Inhibition of MAPK and hypoxic induction of HIF-1α and HIF-2α HT29 pSicoR cells were treated with PD98059 for 6 hours and then incubated in normoxia (N) or hypoxia (H) for 6 hours. Hypoxic induction of HIF-1α and HIF-2α proteins and phosphorylation of p42/p44 (ERK) were analyzed by western blotting. Band densities were quantified and HIF protein levels were normalized to β-actin. Numbers below the bottom panel represent the mean fold change relative to DMSO control for 2 independent experiments. *P<0.05.