Melatonin abrogated p300-augmented COX-2 or iNOS proteins and p52 DNA binding stimulated by LPS/IFNγ. (A-B) RAW 264.7 cells transfected with p300 or control vectors were treated with MT or Trp for 30 minutes followed by LPS or LPS/IFNγ for 8 hours. COX-2, iNOS, and p300 protein levels were analyzed by Western blotting. (C) Cells were cotransfected with p300 and E1A as described in “Materials and methods,” or transfected with p300 followed by treatment with roscovitine (RST, 25 μM) for 30 minutes with or without MT. Binding of nuclear extract p52 to COX-2 or iNOS promoter probe or control probe was analyzed by streptavidin-agarose pulldown assay. (D) Cells were treated by an identical protocol as described in panel C. Chromatin was immunoprecipitated by a p52 antibody (anti-p52) or non–immune IgG (C-IgG). The targeted promoter regions of COX-2 and iNOS were amplified by PCR.