FIGURE 4.

Inhibition of association between NF-κB and κB-binding site of claudin-2 promoter region by AZA and LY-294002. A, wild type (WT) and NF-κB mutant of claudin-2 promoter luciferase vectors were co-transfected with the pRL-TK vector into the cells. At 24 h after transfection, the cells were treated with DMSO vehicle (cont) or 10 μm AZA for 8 h. The promoter activity is represented as a percentage relative to the values in the control of WT. B and C, nuclear proteins were prepared from the control, AZA-, and LY-294002-treated cells. After immunoprecipitation of genomic DNA by anti-NF-κB antibody, semi-quantitative PCR (B) and quantitative real time PCR (C) were performed using the primers amplifying the putative NF-κB-binding site (194S/395A) and 1,000-bp region (8S/179A) upstream from the putative NF-κB-binding site. To confirm that the same amounts of chromatin were used for the immunoprecipitation, input chromatin was also used. The PCR products were visualized with ethidium bromide. The amount of PCR products is represented relative to the value of control cells (194S/395A). n = 3–4. **, p < 0.01 compared with control. NS, p > 0.05 compared with control.