Figure 2.

Overexpression of DDX60 or DDX60L does not induce type I IFNs. (A) Different human DDX60 and DDX60L constructs labeled A to H used in (B) for Western blot analysis and (C) IFN‐β promoter reporter assay. For (B), HEK293 cells were transfected with indicated plasmids and total cells lysates analyzed by Western blot. Membranes were probed with indicated antibodies. MYC‐hRIG‐I transfection was used as a control. In (C) HEK293 cells were cotransfected with an IFN‐β promoter firefly luciferase reporter, renilla luciferase control, and indicated amounts of hDDX60 and/or hDDX60L expression plasmids. Twenty‐four hours later, firefly luciferase (F‐luc) activity was measured and normalized to renilla luciferase (R‐luc) activity and to the water control. Plasmids coding 3xFlag‐hMAVS and 3xFlag‐hRIG‐I were used as positive controls, whereas LacZ was used as a negative one. (D) HEK293 cells were cotransfected with an ISRE promoter firefly luciferase reporter, a Renilla luciferase control, 200 ng/mL of control LacZ or 3xFlag‐hDDX60 expressing plasmid as well as indicated hRIG‐I, hMDA5, hTBK1, or hIRF‐3 expressing plasmids or treated with IFN‐A/D (1000 IU/mL). Twenty‐four hours later, luciferase activity was measured. (E) HEK293 cells were cotransfected with an IFN‐β promoter firefly luciferase reporter, a Renilla luciferase control, and 200 ng/mL of control LacZ or 3xFlag‐hDDX60 expressing plasmid. Twenty‐four hours later, cells were transfected with IVT‐RNA or poly(I:C) or infected with SeV. After overnight culture, luciferase activity was measured. For all experiments, one representative of three independent experimental repeats is shown.