Fig. 3.
HIF2α activation results in increased EGFR mRNA translation. (A) Western blot and RT-PCR analysis of EGFR protein and mRNA levels in the VHL−/− RCC, 786-0, cell line (786-0), and 786-0 cells stably expressing wild-type VHL (786-0 + VHL). (B) Western blot (Left) and RT-PCR (Right) analysis of EGFR protein and mRNA levels in 786-0 cells infected with adenovirus expressing GFP or dnHIF for 24–96 h. (C) Western blot analysis of EGFR protein levels in 786-0 + VHL cells infected with GFP, HIF1α or HIF2α for 72 h. Actin served as a loading control in A–C. (D) EGFR radiolabel incorporation in cells pulsed for the indicated times with [35S]methionine ([35S]Met) and immunoprecipitated with an anti-EGFR antibody. Cells were also labeled and immunoprecipitated with a second EGFR antibody as a specificity control (Right). (E) EGFR [35S]Met incorporation in 786-0 cells infected with GFP or dnHIF for 72 h and labeled for 1 h. (F) EGFR [35S]Met incorporation in cells labeled for 2 h and cold-chased for indicated times. Whole-cell lysates (WCL) served as radiolabel incorporation and loading controls in D–F. (G) RT-PCR analysis of EGFR mRNA levels in polysomal fractions of 786-0 cells infected with GFP or dnHIF for 48 h and subjected to sucrose gradient fractionation. The percentage of total EGFR mRNA in each fraction is plotted.