Activated nuclear NFkappaB (RelA) facilitates C/EBP-ϵ DNA binding to consensus sites. (A) Western-blot analysis of NFkappaB subunits and ATF4 localized in the nucleus of COS1, Jurkat, and KCL22 cells. Antibodies against proteins listed in the right panel were used to probe Western blots derived from nuclear lysates (20 μg per lane) prepared from COS1, Jurkat, and KCL22 cells. Equal gel loading controls were Ponceau stained after transfer. (B) Gel shift analysis was performed on nuclear lysates derived from the Jurkat cells transfected with either the wild-type C/EBP-ϵ (C/EBPϵ75T) or mutant C/EBPϵ (75D or 75A) expression constructs. In the right lane, the nuclear lysate was derived from Jurkat cells transfected with C/EBPϵ75D and activated with TPA. C/EBP-ϵ–specific bands are indicated by double arrows. (C) Gel shift analysis was performed as shown in panel B, except that RelA-containing COS1 cells were used for transfection instead of Jurkat cells. In the right lane, nuclear lysate derived from C/EBP-ϵ75D–transfected cells was cleared of endogenous RelA by immunoprecipitation with a specific antibody.