IRF5 specifically interacts with RelA. (A) HEK-293–TLR4-Md2/CD14 cells were transfected with human IRF5 tagged with ONE-strep–tag (N-terminus) and HA-tag (C-terminus; lanes 1 and 3) or an empty vector pBent (lanes 2 and 4) and fixed with formaldehyde. Cross-links were reversed by heating and immunoblotted for bait IRF5 (anti-HA antibodies), or NF-κB subunits and tubulin. (B) HEK-293–TLR4-Md2/CD14 cells were transfected with RelA-FLAG (lane 1) or BAP-FLAG (lane 2). Cell lysates were immunoprecipated with M2 anti-FLAG Sepharose and immunobloted for bait RelA (anti-FLAG antibodies) or IRF5. (C) MDDCs were stimulated with LPS for 1 hour or left untreated. The endogenous interaction between RelA and IRF5 was examined by immunoprecipitation (IP) with anti-IRF5 antibody and immunoblotting with anti-RelA antibody. −AB indicates a mock IP. (D) Nuclear pellet from Triton-extracted HEK-293–TLR4-Md2/CD14 cells was solubilized with DNaseI, and endogenous interaction between RelA and IRF5 was examined after IP as in panel C.