(A) Schematic of DHX29 truncated forms (top panel). 293T cells were transfected with Flag-MDA5 and full-length HA-DHX29 (FL-DHX29), HA-DHX29a, HA-DHX29b, HA-DHX29c, or HA-DHX29d plasmids. Immunoprecipitation with anti-HA beads and immunoblotting with anti-HA and anti-Flag antibodies were performed (bottom panel). (B) 293T cells were cotransfected with ISRE-luciferase (Luc) and FL-DHX29, empty vector (vec), DHX29a, DHX29b, DHX29c, or DHX29d plasmids and stimulated with IC HMW Poly(I:C). ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold increase relative to stimulated FL-DHX29 control cells. (C) Cell lysates were prepared from 293T cells transfected with full-length HA-DHX29 or HA-DHX29a or HA-DHX29c alone or in combination with Flag-MDA5, and then incubated with biotin-labeled HMW Poly(I:C) for 4 h. Immunoprecipitation was performed with anti-biotin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (D) Purified HA-DHX29 in the presence or absence of Flag-MDA5 was incubated with biotin-labeled LMW Poly(I:C) or HMW Poly(I:C) for 4 h. Pull-down experiments were performed with NeutrAvidin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (E) Purified Flag-MDA5 in the presence or absence of BSA (control), FL-DHX29, DHX29a, DHX29b, DHX29c, or DHX29d protein was incubated with HMW Poly(I:C) for 4 h. Pull-down experiments were performed with anti-biotin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (F) Schematic of DHX29 mutations in walker A (WAM-DHX29) and walker B (WBM-DHX29) sites. Purified Flag-MDA5 in the presence or absence of BSA (control), HA-DHX29, HA-WAM-DHX29, HA-WBM-DHX29 protein was incubated with HMW Poly(I:C) for 4 h. Immunoprecipitation with NeutrAvidin beads and immunoblotting with anti-Flag or anti-HA antibody were performed. (G) 293T cells co-transfected with ISRE-Luc and BSA (control), HA-DHX29, WAM-DHX29, or WBM-DHX29 were stimulated with IC HMW Poly(I:C). ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold decrease relative to stimulated FL-DHX29 control cells. Data from (B and G) are representative of three independent experiments and plotted as the mean ± s.d. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant.