(A) Whole cell lysates (WCL) obtained from 293T cells cotransfected with HA-DHX29 and Flag-RIG-I, Flag-MDA5, or Flag-MAVS plasmids were immunoprecipitated with anti-HA beads. The immunoprecipitated product was immunoblotted with anti-Flag antibody. (B) Luciferase activity of 293T cells transfected with an ISRE (upper panel) or NF-ĸB (bottom panel) luciferase reporter, together with vector for RIG-I, MDA5, MAVS, TBK1, along with empty vector (no wedge) or with increasing amounts (wedge) of expression vector for DHX29. (C and D) Coimmunoprecipitation assays were performed on WCL obtained from THP-1 cells infected with HMW Poly(I:C) (C) or EMCV (MOI 10) (D) at the indicated time points. Immunoprecipitation with anti-DHX29 antibody and immunoblotting with anti-MDA5, phosphorylated (p)-IRF3, and total IRF3 antibodies were performed. (E) DHX29 stably transfected (mDHX29) MEFs were stimulated with HMW Poly(I:C) at the indicated time points. WCL were immunoprecipitated with anti-Flag beads and immunoblotted with anti-MDA5, p-IRF3, and total IRF3 antibodies. (F) Purified HA-DHX29 and Flag-MDA5 were co-incubated with or without HMW Poly(I:C) for 4 h. Pull-down experiments were performed with anti-Flag beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (G) Confocal imaging analysis of the colocalization of DHX29 (red) and MDA5 (green) with or without HMW Poly(I:C) or EMCV stimulation for 5 h. Scale bar = 10 μm. (H) Time-course fluorescent microscopy analysis of the colocalization of DHX29 (red) and MDA (green) stimulated by HMW Poly(I:C) at indicated time points were recorded. Scale bar = 10 μm. (I) MDA5 deletion constructs were generated from full-length MDA5 (FL-MDA5) (top panel). WCL obtained from 293T cells cotransfected with HA-DHX29 and FL-MDA5, N-MDA5, M-MDA5, or C-MDA5 were immunoprecipitated with anti-Flag beads. The immunoprecipitated product was immunoblotted with anti-HA and anti-Flag antibodies. Data from (B) are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant.