Inhibition of p52 DNA binding by MT. (A-C) RAW 264.7 cells were pretreated with MT (1 mM) for 30 minutes followed by LPS or LPS/IFNγ for 8 hours. Nuclear extracts from cells were incubated with (A) a biotinylated iNOS promoter probe corresponding to sequence + 1 to –1168, (B) a COX-2 promoter probe (–30/–508), or (C) a 22-bp κB-containing probe, a 22-bp κB-mutant probe, and a 22-bp control probe, which does not contain any known enhancer element (please see “Materials and methods” for their sequences). The DNA-protein complexes were pulled down with streptavidin-agarose beads, and p52 in the complex was analyzed by Western blots using a specific antibody. C denotes control probe; iNOS, iNOS promoter probe; COX-2, COX-2 promoter probe; mt-κB, mutant κB probe; and wt-κB, wild-type κB probe. (D) ChIP analysis of p52 binding to a 678-bp COX-2 or 498-bp iNOS promoter region. C-IgG refers to immunoprecipitation of chromatin with a non–immune IgG. Cell treatment protocol was identical to that described in panels A-C. (E) p52 protein levels in nuclear extracts in panels A-C were determined by Western blots. Each figure is representative of 2 to 3 experiments with similar results.