NF-κB pathway activation is enhanced in diffDCs but is not responsible for the unique cytokine profile of diffDCs. (A) imDCs and diffDCs were cultured in medium alone as a control (0) or were stimulated with 500 ng/mL LPS for the indicated times. Whole-cell lysates were then electrophoresed and probed with phospho–NF-κBp65, followed by an appropriate secondary antibody. (B) Degradation of IκB-α and nuclear translocation of NF-κB was enhanced in diffDCs. Cytoplasmic or nuclear extracts from DCs cultured in medium alone or stimulated with 500 ng/mL LPS for 10, 20, 40, or 60 minutes were prepared, blotted, and probed with IκB- and NF-κB–specific antibodies, respectively. (C) NF-κB activation was not required for the IL-10 overexpression and impaired IL-12p70 secretion in diffDCs. DCs were pretreated with 100 μmol/mL PDTC, an inhibitor of NF-κB activation, for 30 minutes, then stimulated with LPS for 24 hours. Levels of IL-10 and IL-12p70 in supernatants were determined by ELISA. *P < .05. NS indicates not significant.