EPO translational derepression in response to hypoxia of HeLa cells is mediated by leaky scanning of ribosomes through the inhibitory uORF. The pGL-WT (construct 1), pGL2-no_STOP (construct 2), and pGL2-optimal_uAUG (construct 3) vectors, represented as in Figure 3, were separately cotransfected with a plasmid encoding RLuc (pRL-TK) in HeLa cells. Six hours after transfection, cells were untreated or treated for 24 h with 200 µM CoCl2 to mimic hypoxic conditions. (A) Representative Western blot analysis of transfected HeLa cell extracts untreated (−) or treated (+) with CoCl2 as shown. Immunoblotting was performed using a human HIF1α–specific antibody to control for hypoxia and a human α-tubulin–specific antibody to control for variations in protein loading. (B) Relative luciferase activity was quantified as described in the legend to Figure 2B.
