NO and hypoxia-enhance HIF-1 binding activity.
(A) Oligonucleotide sequences for EMSA. Nucleotides of functional transcription factor binding sites are underlined, and substituted bases are shown in lowercase letters. WT HIF was used as a labeled probe. (B, C) EMSAs showing the binding specificity of nuclear factors from SNAP-treated cells (B) or hypoxia-treated cells (C). Nuclear extracts (5 μg) from A-172 cells, treated by 0.1% DMSO or 0.5 mmol/L SNAP (B), or under normoxic or hypoxic (1% O2) conditions (C), were incubated with WT HIF probe for 30 minutes in the presence of no competitor0 or 10-, 50-, or 250-fold molar excess of unlabeled competitor oligonucleotides. SNAP-induced or hypoxia-induced (H1 and H2), constitutive (C1, C2, C3), and nonspecific (NS) complexes are indicated. (D) SS of HRE binding complexes. Nuclear extracts (5 μg) from A-172 cells treated by 0.5 mmol/L SNAP or by hypoxia were incubated with labeled WT HIF probe, in the presence or absence of mAbs against HIF-1α, HIF-1β, or c-Myc as potential supershifting reagents. The shifted complexes (SS) are indicated. Ab = antibody, N.E. = nuclear extract, S = SNAP, and H = hypoxia.
