LPS induces the HIF-1 transcription factor complex. Quiescent NR8383 cells were maintained either under control conditions (20.8% oxygen, □), or under hypoxic conditions (1% oxygen, ▦), or in the presence of LPS (1 μg/mL, ▪) for 6 hours followed by preparation of nuclear extracts. (A) Nuclear protein (10 μg) was incubated in a 96-well plate coated with an oligonucleotide containing the wild-type (W26) or mutant (M26) HIF-1-binding site. Presence of HIF-1 transcription complex was evaluated with an antibody to either HIF-1α or HIF-1β. (B) Nuclear protein (10 μg) was incubated in a 96-well plate coated with an oligonucleotide containing the wild-type (W26) in the presence or absence of increasing concentrations of wild-type or mutant competitor oligonucleotide. Presence of HIF-1 transcription complex was evaluated with an antibody to HIF-1α. HIF-1 binding was then revealed by incubation with an HRP-conjugated secondary antibody and substrate. Results are expressed as the fold increase of the absorbance at 450 nM over control conditions. This experiment is an average ± SD of an experiment performed in triplicate and is representative of at least 3 independent experiments.