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. 2019 Jul 24;133(5):jcs230409. doi: 10.1242/jcs.230409

Fig. 5.

Fig. 5.

DDX56 interacts with IRF3. (A) DDX56 interacted with IRF3 but not with IRF7 in the overexpression system. 293T cells (2×106) were transfected with the indicated plasmids (5 μg each). Co-immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. The expression of the transfected proteins was analyzed by immunoblotting with anti-HA or anti-Flag antibodies. (B) Colocalization of DDX56 with the IRF3. HeLa cells were transfected with HA–IRF3 (1 µg) and Flag–DDX56 (1 µg) plasmids. At 24 h after transfection, cells were left untreated or infected with SeV for 6 h before confocal microscopy. Scale bar: 5 µm. (C) Endogenous association between DDX56 and IRF3. 293T cells were left untreated or treated with SeV for the indicated times before co-immunoprecipitation and immunoblot analysis. (D) DDX56 mutants interact with IRF3. 293T cells (2×106) were transfected with the indicated plasmids (5 µg each). Co-immunoprecipitations were performed with anti-Flag or control IgG. Immunoblotting analysis was performed with anti-HA antibody (upper panels). Expression levels of the proteins were analyzed by immunoblotting analysis of the lysates with anti-HA and anti-Flag antibodies (lower panels). (E) The 1–197 domain of IRF3 is required for its interaction with DDX56. Experiments were performed in a similar manner to those in C. (F) The D166 site of DDX56 is required for its interaction with IRF3. Experiments were performed in a similar manner to those in C. (G,H) Effects of DDX56, its mutants and its point mutants on the IFN-β promoter and ISRE activation. 293T cells (105) were transfected with the ISRE (H) or IFN-β promoter (G) luciferase plasmids (0.1 μg) and the indicated expression plasmids (100 ng). At 24 h after transfection, cells were infected with or without SeV for 10 h before the luciferase assays were performed. The experiment shown is representative experiments of three independent experiments with mean±s.d. of three technical replicates. EV, empty vector; Luc, luciferase; LC, light chain; HC, heavy chain.