Figure 3: Brd3 associates with IRF3 and promotes IRF3-mediated IFN-β production.

(a), Brd3-ko cells and control cells were infected with VSV(MOI = 10) for the indicated times. Cells were lysed and subjected to Western blot analysis (40 μg) with the indicated antibodies. (b), Brd3-ko cells and control cells were infected with HSV (MOI = 10), VSV (MOI = 10), SeV (MOI = 10) for 8 hours or stimulated with LPS(100 ng/ml) for 90 minutes. Nuclear proteins were extracted and subjected to Western blot analysi s(25 μg) with the indicated antibodies. (c), RAW264.7 cells were infected with VSV (MOI = 10) for the indicated times. Cells were extracted as cytoplasmic and nuclear proteins and subjected to Western blot analysis (25 μg) with the indicated antibodies. (d), RAW264.7 cells were infected with VSV (MOI = 10) for the indicated times. Cell lysates (500 μg) were immunoprecipitated with Brd3 antibody and then immunoblotted with the indicated antibodies. Similar results were obtained in three independent experiments. (e), HEK293T cells were co-transfected with 100 ng IRF3-wide type expressing plasmid; 50 ng IFN-β luciferase reporter plasmid; and 5 ng pTK-Renilla-luciferase reporter plasmid together with 50, 100, or 150 ng of Brd3-expressing plasmid. Total amounts of plasmid DNA were equalized using an empty control vector. After 24 hours of culture, luciferase activity was measured and normalized by Renilla luciferase activity. (f), HEK293T cells were co-transfected with 50 ng IFN-β luciferase reporter plasmid; 5 ng pTK-Renilla-luciferase reporter plasmid together with 50, 100, or 150 ng of Brd3-expressing plasmid and detected as in (e). Data are shown as means ± SD of three independent experiments. *P < 0.05(ANOVA).