RIP140 regulates proinflammatory gene expression via direct promoter association. (A,B) Transient transfection assay of RAW264.7 cells cotransfected with a luciferase reporter containing − 1080 bp of the TNFα 5′-flanking region and an expression vector for RIP140 or empty pcDNA vector (A) or with plasmids carrying RIP140-specific or nonspecific control shRNA (B). Twenty-four hours after transfection, cells were either left untreated or treated with LPS (1 μg/mL) for 24 hours as indicated. Data are means plus or minus SEM (n = 9). *P < .05. (C) Transient transfection assay of RAW264.7 macrophages cotransfected with TNFα luciferase reporter carrying wild-type (wt) sequence from −121 to −88 bp or mutations in binding sites for Ets (muEts), AP1 (muAP1), or NFκB (muκB), together with an expression vector for RIP140 or empty pcDNA vector. Twenty-four hours after transfection, cells were treated with LPS (1 μg/mL) for 24 hours as indicated. Data are means plus or minus SEM (n = 9). *P < .05. Schematic representations of wild-type and mutated promoter constructs are shown. (D) Chromatin immunoprecipitation (ChIP) assay of RAW264.7 macrophages using antibodies specific for RIP140 (αRIP140) or nonspecific IgG (αHA). Precipitated fragments were analyzed by real-time PCR using IL-1β, IL-6, and GAPDH promoter primers. Data show fold enrichment relative to control IgG. Region 1, including NFκB site; region 2, without NFκB site. Data are means plus or minus SEM (n = 2). *P < .05.