Expression of IKK2dn or NIKdn into DCs does not block LPS- or lipid A–induced NF-κB activation and cytokine production.

Immature DCs were left uninfected (white) or were infected with Adβ-gal (gray), AdNIKdn (cross-hatched), AdIKK2dn (checkered), and AdIκBα (black), and cultured for 2 additional days. (A-C) Cells were stimulated for 24 hours with 100 ng/mL LPS or 1 μg/mL of lipid A, and supernatants collected and assayed for the presence of TNFα, IL-6, and IL-8 by ELISA. Mean values ± SDs of 1 experiment representative of 4 independent experiments performed on samples from from unrelated donors are shown. (D) Cells were stimulated with various concentrations of LPS, and supernatants collected and assayed for the presence of TNFα by ELISA. Mean values ± SDs of 1 experiment representative of 3 independent experiments performed on samples from unrelated donors are shown. (E) After stimulation with 100 ng/mL LPS for 45 minutes, cytosolic and nuclear extracts were collected. The presence of cytosolic IKK2 and IκBα was examined by Western blotting and the nuclear NF-κB DNA binding activity determined by EMSA.