C/EBP-ϵ interacts with p65RelA. (A) Specific binding of GST–C/EBP-ϵ fusion proteins to p65RelA is facilitated by the presence of phosphomimetic Asp75 (D) residue within the p38 MAPkinase target site. Equal quantity (40 μg) of immobilized bacterially produced GST–C/EBP-ϵ or MBP–C/EBP-ϵ protein and its mutant or truncated derivatives and isoforms were incubated with nuclear lysates containing p65RelA. Beads were washed extensively in conditions of either normal salt (150 mM), 50 mM, or 250 mM salt (where indicated by line with arrows). Bacterially produced GST and the GST fusion protein containing the full-length resistin-like hXCP1 protein (GST-XCP) were used as negative controls. Eluted proteins were analyzed by Western blotting using specific anti-p65RelA antibodies (Santa Cruz Biotechnology). (B) C/EBP-ϵ–NFkappaB interaction in nuclear lysates of COS1 cells transfected with expression constructs for p30 and p32 isoforms of C/EBP-ϵ. Immunoprecipitation (IP) was performed with antibodies specific to proteins listed above the lanes. Coimmunoprecipitated C/EBP-ϵ was identified by Western blotting with rabbit polyclonal anti–C/EBP-ϵ antiserum. (C) Endogenous p65RelA is coimmunoprecipitated with C/EBP-ϵ, but not ATF4, in COS1 cells transfected with expression constructs listed below each lane or control pcDNA3.1 expression vector. Following immunoprecipitation with either C/EBP-ϵ– or ATF4-specific antibody, p65RelA was identified by Western blotting.