Binding of NF-κB to the TM promoter. HUVECs were stimulated with or without 80 ng/mL human TNF-α for 1 hour. (A) EMSA was performed on nuclear extracts with probes containing either the sequence of a potential noncanonical NF-κB binding site within the TM promoter (left) or the classic NF-κB consensus sequence (right). Competition with 100-fold excess of unlabeled oligonucleotides failed to eliminate nonspecific DNA-protein complexes (solid arrows) but did reduce intensity of p50/p65-DNA complexes (open arrow) induced by TNF-α. (B) Chromatin immunoprecipitation was performed by using primers/probe specific for either the TM (left) or tissue factor (right) promoters and antibodies specific for various NF-κB subunits. ▪ indicates control; ▦, stimulation with TNF-α. Values are the mean ± SEM for 4 experiments. P values were not significant for paired vehicle and TNF-α groups except where indicated.