Figure 1.

LPS and dsRNA activate IRF-3 and IRF-7. (a) BM-derived macrophages from WT and MyD88-deficient mice were stimulated with LPS (0.1–100 ng/ml), Malp-2 (5 nM), and dsRNA (1–100 μg/ml) for 12 h. The concentration of RANTES was measured by ELISA. (b) Nuclear extracts were isolated from WT and MyD88-deficient macrophages stimulated with LPS (10 ng/ml), Malp-2 (5 nM), and dsRNA (50 μg/ml) for 1 h and subjected to EMSA using a ³²P-labeled ISRE consensus sequence (ISG-15) as a probe. Activated complexes were visualized by autoradiography. Activated ISRE DNA-binding complexes were preincubated with polyclonal antibody to IRF-3 or two control antibodies before incubation with the ISRE probe (right). (c) TLR3 and TLR4/MD2-expressing HEK293 cell lines were transfected with a luciferase reporter gene containing the Gal4 upstream activation sequence and with Gal4-DBD, Gal4–IRF-3, or Gal4–IRF-7 (40 ng). After 24 h, cells were stimulated with LPS (10 ng/ml), dsRNA (50 μg poly IC/ml), IL-1β (10 ng/ml), or left untreated for ∼8 h, and luciferase reporter gene activity was measured.