EMSA with the HREs of the human PAI-1 promotor.

Oligonucleotides corresponding to wild-type (wt) or mutated (mut) HREs (sequences are listed in Table 1) were incubated with either nuclear extracts (NE) from normoxic cells (N) or hypoxic cells (H) or from in vitro–translated proteins. (A) NE from H and N cells was incubated with wt and mut probes corresponding to all 5 putative HRE elements of human PAI-1. The constitutive complexes are marked with a C, and the hypoxia-induced complex with an I. (B) For competition assay, radiolabeled wt HRE-2 probe was incubated with NE from hypoxic cells with cold wt or mut probe in 5-, 50-, or 500-fold molar excess, as indicated. (C) Radiolabeled wt HRE-2 probe was incubated with either in vitro–translated HIF-1, ARNT, or both. Rabbit reticulocyte lysate was used as control. (D) For supershift analysis, the wt HRE-2 probe was incubated with NE from H or N cells, as indicated. After initial binding, the preimmune serum, anti–HIF-1 antibody, or anti-ARNT antibody was added. The constitutive complexes are marked C, the inducible I, and the supershifted S.