FIGURE 5.

WT1 does not stabilize HIF-1. A, chromatin was immunoprecipitated from MHH-ES cells grown under normoxia or hypoxia (1% O₂) as well as from SK-ES-1 cells transfected with an empty expression vector (pCB6; labeled “Control”) or a pCB6 expression vector containing cDNA for the indicated WT1 isoform using antibody against HIF-1α or a control IgG. DNA was recovered from the immunoprecipitated chromatin and analyzed by quantitative PCR using primers surrounding the HRE in the VEGF promoter. The graph shows the relative enrichment of HRE in the HIF-1α immunoprecipitates compared with IgG control. Error bars represent S.E. of experiments done in triplicate. Experiments were repeated a minimum of three times. B, RNA was isolated from the same SK-ES-1 cells used for the experiment in A and reverse transcribed. RT-PCR was performed using primers that amplify WT1 (top) and the ribosomal RNA 36B4 (bottom). This experiment was repeated three times.