PMA, IL-1β, and LPS increased p300 and PCAF binding without altering their protein levels. (A) Western blot analysis of p300, CBP, and PCAF protein levels in nuclear extracts prepared from HFb treated with or without PMA (100 nM), IL-1β (10 ng/mL), or LPS (2 μg/mL) for 4 hours. General transcription factor TFII_B was used as a nuclear protein control. (B) Binding of p300, CBP, and PCAF to a COX-2 promoter probe. Nuclear extracts prepared from HFb were incubated with a 424-bp biotinylated COX-2 promoter probe (–30 to –453) and streptavidin-agarose beads for one hour, and the complex was obtained after centrifugation. Proteins in the complex were resolved by Western blot analysis. Co indicates a control nonrelevant probe; and COX-2, the COX-2 probe. (C) Correlation of transactivator binding with p300 binding. Binding assays were performed as described in panel B. Multiple proteins were simultaneously analyzed in the pulldown complex. (D) Binding of coactivators and p300 to chromatin COX-2 promoter. ChIP assays were performed as described in “Materials and methods.” DNA input denotes the loading of an equal amount of nuclear extract DNA without immunoprecipitation. Control IgG indicates rabbit normal IgG. (E) Interaction between p300 and transactivators. Nuclear extracts from HFb treated with or without PMA (100 nM) for 4 hours were immunoprecipitated with each indicated antibody, and p300 in the complex was detected by Western blots. IP indicates immunoprecipitation; and control IgG, rabbit normal IgG. In all panels, the top panel shows a representative of 3 experiments and the bottom panel shows mean ± SEM of densitometry from 3 experiments.