Fig. 4.

RSG attenuates HYP-induced HYP-inducible factor (HIF)-1α and nuclear factor (NF)-κB nuclear binding in HPAEC. HPAEC were exposed to NOR (21% O₂) or HYP (1% O₂) for 72 h. During the final 24 h of exposure, selected cells were treated with RSG (RSG, 10 μM). Nuclear proteins were extracted from HPAECs and then incubated with radiolabeled HIF-1α (A) and NF-κB (C) oligonucleotides and subjected to electrophoretic mobility shift assay analysis. DNA-protein complexes were separated on a native polyacrylamide gel, and densitometric analysis of bands was performed (B and D). Arrows, probe shift due to HIF-1α and NF-κB protein binding and unbound free probe; P, probe alone; C, control 50× unlabeled probe; Cm, control mutated 50× probe. Each bar represents the mean ± SE. P < 0.05 vs. NOR (*) and vs. HYP (+); n = 3. In E, the intracellular localization of the NF-κB subunit p65 was investigated by immunofluorescence by using specific p65 antibody. Images are representative of 3 experiments. Scale bar = 10 μm. There was no staining with nonimmune IgG isotype control primary antibody (data not shown).