Fas ligation activation of NF-κB in DCs and its involvement in DC survival. (A) Determination of Fas ligation-induced phosphorylation of IκB and nuclear translocation of NF-κB. Cytoplasmic or nuclear extracts from DCs cultured in medium alone (C) or stimulated with Jo-2 (J), isotype antibody (I), or LPS (L) were prepared, blotted, and probed with phospho-IκB- and NF-κB-specific antibodies, respectively. (B) DCs were pretreated with different doses of PDTC, an inhibitor of NF-κB activation, for 30 minutes, then stimulated with Jo-2, isotype antibody or LPS, for 24 hours. Levels of IL-1β in supernatants were determined by ELISA, and DCs were harvested for CD86 staining and analysis by flow cytometry. (C) Detection of Fas ligation-induced DC death. After pretreatment with the indicated dose of PDTC, DCs were stimulated with Jo-2 or isotype antibody for 8 hours, then stained with R123 and analyzed by flow cytometry. Data are presented as the percentage of cells that were apoptotic (R123low).
