Fig. 5.

KDM2B interacts with Brg1 to increase the chromatin accessibility of the Il6 promoter. a, b Immunoblot analysis of KDM2B and Brg1 in protein complexes immunoprecipitated with an anti-KDM2B antibody (a) or anti-Brg1 antibody (b) from lysates of peritoneal macrophages stimulated with LPS for the indicated times. c, d HEK 293T cells were cotransfected with Flag-Brg1 and V5-KDM2B-WT (wild type), V5-KDM2B-ΔJmjc (Jmjc domain deletion), V5-KDM2B-ΔCXXC (CXXC domain deletion), V5-KDM2B-ΔPHD (PHD domain deletion), V5-KDM2B-ΔFBOX (F-box domain deletion), or V5-KDM2B-ΔLRR (LRR domain deletion), followed by immunoprecipitation with an anti-V5 antibody (c) or anti-Flag antibody (d) and immunoblot analysis with an anti-Flag antibody (c) or anti-V5 antibody (d). e Re-ChIP-PCR assay of KDM2B-Brg1 interaction at the Il6 promoter in peritoneal macrophages stimulated with LPS for 1 h. First-round ChIP (1st antibody) was performed with an anti-KDM2B antibody or IgG. Eluted samples were subjected to second-round ChIP (2nd antibody). Line 5 was the input. f ChIP-qPCR analysis of Brg1 occupancy at the Il6 promoter in Kdm2b^−/− (KO) and Kdm2b^+/+ (WT) peritoneal macrophages stimulated with LPS or poly(I:C) for the indicated times. g Chromatin accessibility of the Il6 promoter region assayed by DNase I sensitivity analysis in macrophages transfected with Brg1 siRNA (Brg1 siRNA-1) or control siRNA (Ctrl siRNA-1) and, 48 h later, stimulated with LPS for the indicated times. h, i Q-PCR analysis of Il6 mRNA expression and ELISA of IL-6 production in supernatants of peritoneal macrophages transfected with Brg1 siRNA (Brg1 siRNA-1) or control siRNA (Ctrl siRNA-1) and, 48 h later, stimulated with LPS (h) or poly(I:C) (i) for the indicated times. The data are from three independent experiments (f–i, mean ± s.e.m.) or are representative of three independent experiments with similar results (a–e). *P < 0.05; **P < 0.01