Ddx25

For luciferase reporter assays, 70% confluent HEK293T cells were transfected with 10 ng of pRL-TK reporter plasmid (herpes simplex virus thymidine kinase promoter driving Renilla luciferase, internal control), 100 ng of IFNβ luciferase reporter plasmid (firefly luciferase, experimental reporter), 50 ng of IFNβ activators (RIG-IN, MAVS, TBK1, IKKε, IKKα, NFκB, or IRF3), as well as either 100 ng of recombinant expressing plasmids or siRNAs [Vector, DDX25, 50 nM negative control (N.C.), or DDX25 siRNA]. For measuring the activation of transcription factor NFκB and IRF3, NFκB and IRF3 responsive element specific reporter plasmids were used in the luciferase reporter assays. Subconfluent HEK293T cells were transfected with 10 ng of pRL-TK reporter, 100 ng of NFκB (pNFκB-Luc), or IRF3 (pPRD(III–I)-Luc) luciferase reporter plasmid, various doses of recombinant expression plasmids (Vector or DDX25), along with 50 ng of expression plasmids of RIG-IN. At 24 h post-transfection, cells were infected with DENV-2 at an MOI of 1 and incubated further for 24 h. Luciferase activity was measured using a Promega Dual Glow Kit according to the instructions of the manufacturer (Promega, USA).