(A) WCL obtained from 293T cells co-transfected with Flag-tagged full-length (FL)-MDA5, N-MDA5, M-MDA5, or C-MDA5 and HA-DHX29a were immunoprecipitated with anti-Flag beads. The immunoprecipitated product was immunoblotted with anti-HA and anti-Flag antibodies. (B) 293T cells were cotransfected with ISRE-luciferase (Luc) and FL-DHX29, empty vector (vec), DHX29a, DHX29b, DHX29c, or DHX29d plasmids and stimulated by overexpressing MDA5. 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) 293T cells transfected with Flag-MAVS, HA-DHX29, Flag-MDA5, or Flag-MDA5 plus HA-DHX29 were incubated with biotin-labeled LMW Poly(I:C) for 4 h. (D) ISRE-luciferase (Luc) activity in 293T cells transfected with DHX29 and increasing concentrations (0, 100, and 200 ng per well) of WAM-DHX29, WBM-DHX29, DHX29a, or DHX29b and stimulated with intracellular (IC) HMW Poly(I:C). ISRE-Luc activity is expressed as the fold increase relative to the unstimulated control. (E, F) 20ng Flag-RIG-I (E) or Flag-MDA5 (F) transfected 293T cells were co-transfected with wildtype DHX29, DHX29c, WAM and WBM of DHX29, followed by stimulation for 6 hrs or not. The lysates were immunoprecipitated with anti-Flag beads and immunoblotted with indicated antibodies. Cal A was added 1hr before lysate collection. Data from (B, D) are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. IC Poly(I:C)-stimulated cells (two-tailed Student's t-test). Related to Fig 6 in the main text.