Figure 3.

The high-altitude deer mouse Hif-2α T755M mutation impairs transcriptional activity.A, Left, HEK293FT cells were transfected, and equal protein extract amounts examined by Western blotting. Right, cells were transfected 10 ng of (eHRE)₃-Luc, 10 ng of RL-TK, and 30 ng of plasmids expressing the indicated proteins. WT indicates that the amino acid at residue 755 is Thr. Twenty hours after transfection, luciferase activities were measured. B, Left, HEK293FT cells were transfected with plasmids expressing GAL4 or either WT or T755M HA-GAL4-Hif-2α (669–874). Equal protein extract amounts were examined by Western blotting. Right, cells were transfected 20 ng of (GAL4)₅-E1b-Luc, 20 ng of RL-TK, and 30 ng of plasmids expressing the indicated proteins. Four hr after transfection, cells were exposed to 0.5% O₂ or maintained under normoxia for an additional 18 h and luciferase activities measured. C, Flp-In TRex 293 cells stably transfected with the indicated constructs or parental Flp-In TRex 293 cells (Cont) were induced with tetracycline. Equal protein extract amounts were examined by Western blotting. D–H, RNA was harvested from these cells, reverse transcribed, and then real-time PCR performed to measure the transcript levels from (D) the deer mouse Hif2a transgene, and the genes for (E) vascular endothelial growth factor A (VEGFA), (F) N-myc downstream regulated gene 1 (NDRG1), (G) adrenomedullin (ADM), and (H) solute carrier family 7 member 5 (SLC7A5). I, HEK293FT cells were transfected, flag-tagged proteins were immunoprecipitated, and Western blotting was performed. In panels A, B and D–H, ∗∗p < 0.01, ∗p < 0.05, and ns, not significant by Student's t test. The means ± SD are shown. Hif-2α, hypoxia-inducible factor-2α.