LPS stimulates nuclear translocation and recruitment of transcriptional activators to the CD40 promoter and histone modifications. (A) RAW264.7 cells were incubated in the absence or presence of LPS (10 ng/mL) for up to 4 hours. Cytoplasmic and nuclear fractions were prepared and assayed with anti–phospho-NF-κB p65^Ser536 and anti–phospho-STAT-1α^Tyr701. The cytoplasmic and nuclear fractions were also probed with anti–caspase-3, anti–c-Jun, anti–NF-κB p65, or anti–STAT-1α antibodies. (B) RAW264.7 cells were treated with LPS (10 ng/mL) for up to 4 hours, then the cells were crosslinked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with anti–NF-κB p65, anti–NF-κB p50, anti–STAT-1α, and anti–Pol II antibody, or normal rabbit IgG. The basal level of the untreated sample was set as 1.0, and fold induction on LPS treatment was compared with that. Representative of 3 to 4 experiments. (C) RAW264.7 cells were treated with LPS (10 ng/mL) for up to 4 hours, then the cells were crosslinked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against histone acetylation, methylation, and phosphorylation. Fold induction on LPS treatment was calculated as in panel B. Representative of 3 experiments. (D) EOC13 cells were treated with LPS (10 ng/mL) for up to 4 hours, then the cells were crosslinked with formaldehyde. Soluble chromatin was subjected to immunoprecipitation with antibodies against NF-κB p65, anti–NF-κB p50, STAT-1α, Ac-H3, and Ac-H4. Fold induction on LPS treatment was calculated as in panel B. Representative of 3 experiments.