RelA is required for IRF5-mediated activation of TNF. (A-C) HEK-293–TLR4-Md2/CD14 cells were transfected with siRNA against RelA (siRelA) or with nontargeting siRNA (siC) and used in ChIP analysis of RelA and IRF5 recruitment. Data indicate mean percentage input relative to gDNA ± SD of a representative experiment. −AB indicates a no-antibody control. (A) A total of 75% of RelA protein was degraded estimated by serial dilutions of the siC control sample analyzed by Western blotting. (B) Reduction in LPS-induced RelA recruitment to region H in siRelA-treated cells. (C) Reduction in LPS-induced IRF5 recruitment to region H in siRelA-treated cells. (D) HEK-293–TLR4-Md2/CD14 cells were transfected with the RelA, IRF5, and MyD88 expression constructs together with the TNF 5′ upstream/luciferase/TNF 3′ downstream reporter plasmids: 5′wt/3′wt indicates wild-type construct; 5′mut/3′wt indicates mutated κB2 (GTGAATTCCC → tTGAATTCCC), κBξ (GTGATTTCAC → aTccTTTCAC), and κB2a (GGGCTGTCCC → taGCTGTGCCC) sites in the TNF 5′ upstream; 5′wt/3′mut indicates mutated κB4 (GGGAATTTCC → cGcAATgTgC) and κB4a (GGGAATTCCA → cGcAAgTgCA) sites in the TNF 3′ downstream; and 5′mut/3′mut indicates all κB sites mutated. Data show means ± SD and are a representative of 3 independent experiments, each performed in triplicate.