FIGURE 4.

NF-κB p65 binds to the p19 promoter region. A, LPS induces NF-κB DNA complex formation. RAW264.7 cells were activated with IFN-γ (10 ng/ml), LPS (1 μg/ml), or LPS plus IFN-γ for 4 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were added to the ³²P-labeled probe (−228 to −198 of the p19 promoter). Extract and probe were incubated with 0.5 μg of poly(dI-dC) at room temperature for 30 min. B, p65 antibody attenuates DNA-protein complex formation. RAW264.7 cells were activated with LPS (1 μg/ml) for 4 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were incubated with 2 μg of p65, p50, or c-Rel antibody and the ³²P-labeled probe used in A. C, chromatin co-immunoprecipitation assays. RAW264.7 cells were activated with LPS for 4 h. Formaldehyde (1% final concentration) was added for 30 min at room temperature. Cells were harvested, and the chromatin was sheared by sonication and purified by immunoprecipitation (IP) with an anti-p65 antibody. After reversal of cross-linking, DNA was amplified by PCR with primers spanning the p19 promoter. D, TLR ligands induce an NF-κB DNA complex. RAW264.7 cells were activated with LPS, PGN, and poly(I-C) for 4 h, and nuclear protein was extracted for EMSA. 5 μg of nuclear protein were added to the ³²P-labeled probe used in A. con, control.