A) HEK293 cell lines were retrovirally infected to stably produce HA-tagged wild-type or the various HIF2A mutants (Pro531Thr=531T, Pro531Leu=531L, Pro531Ser=531S, Ser71Tyr=S71Y) or wild-type (531P) as indicated. The Pro531Ala (P531A) mutant construct is included as a positive control (Kondo et al. 2002). Cell extracts were prepared after cycloheximide exposure for the indicated times and immunoblotted with anti-HA antibody or beta-actin as a loading control. B) Transcript levels of VEGFA, POU5F1(OCT-4) and SLC2A1(GLUT1) of HEK293 cells stably expressing the indicated HIF2A mutants and controls, measured by quantitative real time PCR (qRT-PCR). Results are shown as the average and bars represent standard error of the means. Values were normalized to those of EV control (*indicates statistical significance at p <0.05; **indicates statistical significance at p<0.005 between cells expressing each HIF2A construct vs. EV). C) HEK293 cells expressing HIF2A mutants or controls as above (empty vector=EV) were incubated overnight under normoxia or 1% O2, as shown. Cell extracts were prepared and probed with anti-HA, anti-GLUT1 or a loading control, beta-actin. D) Transcript levels of VEGFA, POU5F1(OCT-4) and SLC2A1(GLUT1) in VHL-reconstituted 786-0 cells stably expressing the indicated HIF2A mutants and controls, measured by qRT-PCR. Results were normalized to those of EV control (*indicates non-significance; all remaining values showed statistical significance at p<0.005 between cells expressing each HIF2A construct vs. EV). E) 786-0 renal carcinoma cells stably expressing the designated 531 HIF2A mutants were reconstituted with an MSCV-HA-VHL retroviral construct or control. Cell extracts were prepared and immunoblotted with GLUT1, HA and beta actin as a loading control.