Identification of NF-κB as a transcription factor in Et-1 activation. Nuclear extracts were prepared from CNS-EC incubated in the absence (lanes 1, 2, and 3) or the presence of Et-1 (10−8 mol/L; lanes 4, 5, and 6) or TNF (10 pg/mL; lanes 7, 8, and 9) for 1 hour. A radiolabeled probe was incubated with nuclear extract (10 μg of protein) for 30 minutes and protein-DNA complexed was resolved by electrophoresis. Specific protein-DNA complexes are indicated by arrows. Nuclear extract from lanes 1, 4, and 7 were analyzed for binding activity. Lanes 2, 5, and 8 contained a 100-fold molar excess of unlabeled double-stranded oligonucleotide probe as a competitor. Lanes 3, 6, and 9 contained a 100-fold molar excess of unlabeled nonspecific probe as competitor. The data presented are from 1 of 3 representative experiments.
