Binding of USF-2a to the rat PAI-1 HREs.

(A) Oligonucleotides. The E-box consensus sequence CACGTG and the sense strands of the rat PAI-1 promoter sequence oligonucleotides containing HRE-2 and HRE-1 are shown. Bases matching the consensus sequences are underlined. (B) EMSA. The ³²P-labeled PAI-1 HRE-1 (−182/−166) (left panel) and HRE-2 (−168/−152) (right panel) oligonucleotides were incubated with either 10 μg protein of nuclear extracts from normoxic (16% O₂) or hypoxic (8% O₂) cells as indicated (see “Materials and methods”). In EMSA with antibodies the nuclear extracts were preincubated with 1 μL preimmune serum, the USF-1, USF-2, Myc, Max, or SP-1 antibodies for 2 hours at 4°C before adding the labeled probe. The DNA protein binding was analyzed by electrophoresis on 5% native polyacrylamide gels. (C) EMSA competition assays. The ³²P-labeled HRE-1 or HRE-2 oligonucleotide was incubated with nuclear extracts as in panel B. For competition, increasing concentrations, as indicated, of nonlabeled HRE-1 or HRE-2 oligonucleotides were added. C indicates constitutive complex; I, induced HIF-1 complex; S, supershifted USF complex.