Figure 3. Impaired activation of IKKα/β and IRF-1 in TLR7/9-stimulated btk^−/− cDC.

Wild type and btk^−/− cDC were stimulated with 1 µM R848 (A) or 0.1 µM CpG (B) for various times as indicated and IKKα/β activation was examined via immunoblot analyses using anti-phospho-IKKα (Ser¹⁸⁰)/IKKβ (Ser¹⁸¹) antibody. The anti-IKKα/β blot was included as loading control. Densitometric ratios of phospho-IKKα (Ser¹⁸⁰)/IKKβ (Ser¹⁸¹) over IKKα/β are graphed and shown in the lower panels of A and B. *p<0.05, **p<0.005 (Student's t test). (C & D) Nuclear translocation of IRF-1 in cDC that were stimulated for 1 h with R848- (C) or CpG- (D). Nuclear extracts were obtained from non-treated or 1 h stimulated wild type and btk^−/− cDC and examined for the presence of IRF-1 via western blot analyses. The anti-HDAC1 blot was included as loading control. The ratio of densitometric values of IRF-1 over HDAC1 is shown in lower panels of C and D. Numbers represent mean±SEM of three experiments. **p<0.005 (Student's t test). (E) Immunofluorescence confocal studies of IRF-1 localization in wild type and btk^−/− cDC that were untreated or stimulated for 1 h with 1 µM R848. Bar = 10 µM. Results shown are representative of at least three independent experiments.