FIGURE 6.

Hypoxia induces the binding of HIF-1 to an HRE in the FBLN5 promoter. HeLa cells were subjected to hypoxic or normoxic conditions or co-transfected with a HIF-1α expression vector (pHIF-1α) or the empty vector (pcDNA3) and nuclear extracts were obtained. A, nuclear levels of HIF-1α were determined by Western blot. Nucleolin levels were used as a loading control. B, the DNA binding activity of HIF-1α was evaluated by EMSA using an oligonucleotide probe corresponding to the putative HRE binding site in the FBLN5 promoter (FBLN5-HRE). The binding to this motif was prevented when the HRE site was mutated (FBLN5mut-HRE). Upon addition of a specific antibody against HIF-1α (Ab-HIF-1α), the intensity of the shifted band was strongly reduced. A nonspecific IgG was used as a control. The arrowheads indicate the position of the specific retarded band and the excess of free probe. A representative autoradiogram of two independent experiments is shown. NE, nuclear extracts.