(A) 293T cells were cotransfected with HA-mouse (m)DHX29 and Flag-mRIG-I, Flag-mMDA5, or Flag-mMAVS. Whole cell lysates (WCL) were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA or anti-Flag antibodies. (B) Scrambled or DHX29 siRNA transfected WT MEF and MAVS knockout MEF were stimulated with Flag-TBK1 overnight. The WCL were subjected to immunoblot with indicated antibodies. (C) Luciferase assay of RIG-I knockout 293T cell transfected with increase amount of DHX29, followed by stimulation of intracellular (IC) LMW Poly(I:C), HMW Poly(I:C), MDA5 or MAVS. (D) RIG-I Knockout 293T cells were cotransfected with HA-DHX29 and Flag-MAVS or Flag-MDA5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-HA or anti-Flag antibodies. Anti-RIG-I antibody is used to verify the RIG-I knockout cell in whole cell lysate. (E) HA-DHX29- and Flag-MDA5 (20 ng)-transfected 293T cells were stimulated with intracellular (IC) HMW Poly(I:C) at the indicated time points. WCL were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA, phosphorylated (p)-IRF3, and IRF3 antibodies. (F) WCL obtained from THP-1 cells stimulated with IC HMW Poly(I:C) at the indicated time points were immunoprecipitated with anti-DHX29 antibody and immunoblotted with MDA5, p-IRF3, and IRF3 antibodies. (G) 293T cells transfected with HA-DHX29 and Flag-RIG-I or Flag-MDA5 were infected with indicated kind of stimulation at 8hr. The cell lysate was immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibodies. (H) WCL obtained from 293T cells transfected with HA-DHX29 and Flag-LGP2 after 6hr EMCV treatment were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA and anti-Flag antibodies. (I) IFN-β- Luc activities in 293T cells transfected with indicated plasmids post HMW Poly(I:C) treatment were determined. (J) 293T cells expressing Flag-MAVS and Flag-MDA5 were transfected with DHX29 siRNA or scrambled (Scr) siRNA and then stimulated with IC HMW Poly(I:C). WCL were immunoprecipitated with anti-HA antibody and immunoblotted with anti-HA, anti-Flag, p-IRF3, and IRF3 antibodies. WCL were immunoprecipitated with anti-biotin beads and immunoblotted with anti-Flag and anti-HA antibodies. Data from (C, I) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to Fig 5 in the main text.