Figure 6. Recruitment of IκBβ to the IL-1β promoter in complex with NF-κB p65/c-Rel. (A) ChIP in RAW264.7 macrophages. RAW264.7 macrophages were stimulated for 2 h with LPS (100 ng/ml). Chromatin was immunoprecipitated with IκBβ-, RelA/p65-, NF-κB1/p50–, NF-κB2/p52–, c-Rel-, and RNA-polymerase II–specific antibodies or IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for IL-1β or MIP-2 promoters. Three independent experiments revealed similar results. (B) ChIP in BMDMs. After stimulation with LPS (100 ng/ml) for 1 h, chromatin was immunoprecipitated with IκBβ-, RelA/p65-, NF-κB1/p50–, NF-κB2/p52–, c-Rel-, and RNA-polymerase II–specific antibodies or IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for IL-1β or MIP-2 promoters. Three independent experiments revealed similar results. (C) Biotin-streptavidin pulldown assay with a κB oligonucleotide, corresponding to the proximal κB site of the IL-1β promoter. RAW264.7 macrophages were stimulated with LPS (100 ng/ml) for 2 h. Nuclear and cytoplasmic extracts were incubated with the κB oligonucleotide and pulled down with streptavidin-agarose. Western blot detected RelA/p65, c-Rel, and IκBβ. One out of three independent experiments is shown.
Figure 6.

Recruitment of IκBβ to the IL-1β promoter in complex with NF-κB p65/c-Rel. (A) ChIP in RAW264.7 macrophages. RAW264.7 macrophages were stimulated for 2 h with LPS (100 ng/ml). Chromatin was immunoprecipitated with IκBβ-, RelA/p65-, NF-κB1/p50–, NF-κB2/p52–, c-Rel-, and RNA-polymerase II–specific antibodies or IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for IL-1β or MIP-2 promoters. Three independent experiments revealed similar results. (B) ChIP in BMDMs. After stimulation with LPS (100 ng/ml) for 1 h, chromatin was immunoprecipitated with IκBβ-, RelA/p65-, NF-κB1/p50–, NF-κB2/p52–, c-Rel-, and RNA-polymerase II–specific antibodies or IgG as a negative control. Precipitated DNA or 10% of the chromatin input was amplified with gene-specific primers for IL-1β or MIP-2 promoters. Three independent experiments revealed similar results. (C) Biotin-streptavidin pulldown assay with a κB oligonucleotide, corresponding to the proximal κB site of the IL-1β promoter. RAW264.7 macrophages were stimulated with LPS (100 ng/ml) for 2 h. Nuclear and cytoplasmic extracts were incubated with the κB oligonucleotide and pulled down with streptavidin-agarose. Western blot detected RelA/p65, c-Rel, and IκBβ. One out of three independent experiments is shown.

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