Gel-shift analysis with nuclear extracts from Hepa-1 cells exposed to normoxia (lane 1) or hypoxia (3% O₂ for 4 hours; lanes 2-6). HIF-1 DNA binding activity was examined with 3 different oligonucleotides containing either 2 (TfHBSww) or 1 (EpoWT) consensus HBSs or a mutated HBS (EpoMut). For confirmation of specificity, competition experiments were performed using a 250-fold molar excess of unlabeled annealed TfHBSww oligonucleotide in the binding reaction. HIF, inducible HIF-1 binding; c, constitutive binding; n.s., nonspecific binding.