Figure 5

Identification of TNFα-responsive region in the P2-COMT promoter. (A) Schematic diagram of the P2-COMT/Luc construct and its serial deletions Del.1 and Del.2. The effect of TNFα (20 ng/ml, 8 h of treatment) on the relative activity of the P2-COMT/Luc, Del.1 and Del.2 constructs transfected into H4 (B) and H4 IκBα-SR (C) cells. Data are Means ± SEM. *P < 0.05 different from untreated control. (D) Binding of p65 to a plate-immobilized oligonucleotide containing a κB binding site was measured by TransAM NF-κB assay in the nuclear extracts from H4 cells 30 min post TNFα treatment. This activation was prevented by competition with a wild-type binding control (WT BC) κB consensus oligonucleotide or a wild-type P2-COMT κB consensus oligonucleotide (WT P2). Oligonucleotides with mutated κB binding sites – a mutant binding control (MT BC) and a mutant P2-COMT (MT P2-COMT) – had little or no effect. Data are Means ± SEM. ^### P < 0.001 TNFα-treated different from untreated control, ***P < 0.001 TNFα-treated different from TNFα+WT BC and WT P2 oligos, *P < 0.05 TNFα-treated different from TNFα+MT BC oligo.