Figure 5.

TAK1-mediated IKKα/β activation is required for IRF3-dependent IFNβ expression in DC. BMDC were stimulated with LPS or dsRNA (p(I:C); 25 μg/ml) in the presence of 1 μM 5Z-7-oxozeaenol (5Z), 2 μM MRT67307 (MRT), or vehicle alone (CTL). (A, B) Immunoblot (IB) analysis of IKKα/β (pIKKα/β; 85 kDa/89 kDa) and IRF3 phosphorylation (Ser396; pIRF3), βactin and IRF3 expression were used as loading controls. (C, D) BMDC were treated as above in the presence or absence of 1 μM MLN4926 (MLN), RNA was extracted at the indicated time points for qRT–PCR analysis of IFNβ mRNA expression; data are represented as fold induction over control, normalized to cyclophillin (CPH) mRNA. (E, F) BMDC were generated from Map3k7^F/F and Map3k7^F/F.CreER mice and treated with 4 μM tamoxifen (OHT) for 48 h, before stimulation with LPS or dsRNA as described above. Protein extracts were prepared at the indicated time points and phosphorylation of IKKα/β and IRF3 was measured by IB analysis; JNK activity was measured by IP kinase assay using recombinant GST-cJun as a substrate (E). In parallel experiments, RNA was extracted at 4 h and IFNβ expression measured by qRT–PCR (F). Representative data from at least two independent experiments are shown. qRT–PCR data are presented as mean±s.e.m. of three replicates.

Source data for this figure is available on the online supplementary information page.

Source Data for Figure 5 ^{(1.2MB, pdf)}