IL-10 inhibits the recruitment of RelA to proinflammatory genes. (A–C) Adenovirus reporters based on the human TNF gene were infected into macrophages and stimulated with LPS with or without IL-10 for 4 h, after which luciferase assays were performed. Cells were either transfected with scrambled control oligonucleotides (siControl) or RelA-specific siRNA (siRelA; A), and cytoplasmic extracts were subjected to Western blotting using anti-RelA or tubulin antibodies to evaluate the extent of RelA knockdown (B) or cells were stimulated for the indicated times with LPS and total RNA was isolated and used as template in a quantitative RT-PCR gene expression assay for TNF mRNA (C). (D) Position of primers used in ChIP assays for the detection of RelA recruitment to the human TNF gene. NF-κB sites are indicated below the diagram, with the PCR targets represented by black lines with the length of the amplicons indicated above. (E) Cells were stimulated with LPS with or without IL-10 for 60 min, after which ChIP assays were performed using RelA-specific antibodies. Data are expressed as mean ± SD of triplicate measurements for a single donor representative of eight (A) or three (C and E) independent experiments using different donors.