Specific binding of C/EBP-ϵ–ATF4 heterodimers to a high-affinity binding site is facilitated by the presence of nuclear RelA. Gel shift analysis was performed using a composite ATF/CEBP binding probe and nuclear lysates from cells transfected with the wild-type C/EBP-ϵ and ATF4 expression vectors. Specific heterodimer binding (indicated by arrows) was confirmed by competition with 5- and 20-fold molar excess of unlabeled self-oligonucleotide and lack of competition with 20-fold molar excess of consensus C/EBP-ϵ or ATF4 homodimer binding oligonucleotides. Specific complexes contain both ATF4 and C/EBP-ϵ, but not p65RelA, as indicated by supershifts produced by addition of specific antibodies (Abs). (A) Nuclear lysates were derived from transfected and TPA- and Ca⁺⁺ ionophore–activated Jurkat cells. (B) Nuclear lysates were derived from transfected COS1 cells. Elimination of endogenous p65RelA from nuclear lysates by preclearing with 10 μg of α-p65RelA per 10 μL of nuclear lysate (second lane from the left) results in disruption of specific binding of ATF4–C/EBP-ϵ heterodimers.