FIGURE 6.
Hypoxia-mediated changes in the translation of HIF-1α, VEGF, and β-actin correlate with changes in mRNA levels under both moderate and severe hypoxia. A and B, Hep3B cells were cultured under normoxic or moderate hypoxic conditions (0.5% O2 42 h) and then metabolically labeled for the last hour with [35S]methionine and [35S]cysteine. The rate of protein synthesis was determined by the incorporation of trichloroacetic acid-precipitable counts. Pulse-labeled Hep3B cells were immunoprecipitated with anti-HIF-1α, anti-p57Kip2, and anti-β-actin antibodies. Cells were pretreated and labeled in the presence of DFX to eliminate the post-translational normoxic degradation of HIF-1α subunits. C, Hep3B cells were cultured under normoxia and prolonged moderate hypoxia, and then 1 h before analysis the media was changed so that only newly synthesized VEFG protein was measured. The concentration of VEGF protein in the media was determined by ELISA assay. D, the levels of HIF-1α, p57Kip2, β-actin, and VEGF mRNA in Hep3B cells cultured under normoxia or prolonged moderate hypoxia were assessed by QRT-PCR. Results are the average of three experiments, and error bars represent the S.E. E, Western blot analysis showing the accumulation of HIF-1α, p57Kip2, β-actin, phosphorylated (P)-eIF-2α, eIF2α, eIF4E, and 4E-BP1 protein under normoxia and prolonged moderate hypoxia. Cells for HIF-1α Western blot analysis were not treated with DFX during culture.