Signaling mature DCs through CD40 induces nuclear translocation of NF-κB, AP-1, and CREB. (A) One hour after incubation with agonist anti-CD40 mAb, mature DCs translocated NF-κB(P = .0004), and to a lower extent AP-1 (P < .0008), into their nuclei. The highest amounts of nuclear translocation factors occurred 3 hours after incubation with the agonist anti-CD40 mAb (P < .0001, P < .0005, and P < .001 for NF-κB, AP-1, and CREB, respectively). Transfection with gene gun did not induce translocation compared with untreated mature DCs, (P > .05) and signaling through CD40 induced nuclear translocation regardless of gene gun transfection (P > .05). Translocation of transcription factors was quantified by ELISA and expressed as -fold of protein increase versus untreated control cells. A value of 1 was set for untreated cells in each situation. Means ± 1 SD from 3 independent experiments are shown. (B) Specific inhibition of NF-κB, AP-1, and CREB resulted in significantly decreased transgene expression by CD40-signaled mature DCs (P < .0001). Controls included transfected mature DCs cultured in medium alone (M) or with anti-CD40 mAb or transfected mature DCs incubated with anti-CD40 mAb in the presence of the serine proteases blocking agent TLCK. Means ± 1 SD from 4 (A) or from 10 (B) independent experiments are shown. P < .0001.