Enhanced binding of HIF-1 to the HREs of the rat PAI-1 promoter because of overexpression of activated PKB.

EMSA: the ³²P-labeled PAI-1 HRE (−182/−157) oligonucleotide was incubated with either 10 μg protein of nuclear extracts from normoxic (16% O₂) or hypoxic (8% O₂) cells transfected with the myrPKB expression vector or the empty control vector, as indicated (see “Materials and methods” for more information). In the EMSA with antibody the nuclear extracts were preincubated with 1 μL HIF-1α antibody for 2 hours at 4°C before adding the labeled probe. The DNA-protein binding was analyzed by electrophoresis on 5% native polyacrylamide gels. I represents induced HIF-1 complex; S, supershifted HIF-1 complex.