IRF-3 activation and binding to IL-12p35 ISRE site in human DCs. (A) DCs were incubated with medium alone, LPS, or PolyI:C for 90 minutes. Subcellular localization of IRF-3 was analyzed by immunofluorescence using anti-IRF-3 (top panels). Nuclei were counterstained with Toto-3 (bottom panels). One representative donor of 3 is shown. (B) Assessment of IRF-3 activation in DCs. DCs were left untreated or stimulated for 90 minutes with LPS or PolyI:C. IRF-3 binding activity in nuclear extracts (5 μg protein) was assessed using an ELISA-based technique (IRF-3 TransAM; Active Motif). Results are presented as mean ± SEM from 3 different donors. OD indicates optical density. (C) Endogenous IRF-3 binds to IL-12p35 ISRE site. DCs were left untreated or stimulated with LPS or PolyI:C for 90 minutes. Nuclear extracts (10 μg protein) were incubated with radiolabeled ISREwt probe from the p35 promoter. When indicated, PolyI:C-treated extracts were incubated with polyclonal IRF-3 antibody or control antibody. To ensure specificity of the binding, nuclear extracts from PolyI:C-stimulated DCs were incubated with radiolabeled ISREwt probe in the presence of a 50-fold molar excess of the indicated unlabeled competitor.