Loss-of-function IFIH1 mutation. (A, C, and D) Relative increase in normalized luciferase activity driven by the IFNΒ1 promoter (A), ISRE (C), or NF-κB (D) reporter constructs. Cells were cotransfected with WT or mutant MDA5, and with (filled) or without (open) transfected poly(I:C) as indicated. R337G and R779H are gain-of-function mutants. (B) Immunoblot for MDA5 proteins after transient transfection of 20 ng WT or mutant K365E MDA5, both under CMV promoters. (E) Similar to A, except that cells were cotransfected with 20 ng WT MDA5 (under CMV promoter) and either K365E MDA5 or empty vector (EV; under MSCV promoter). (F) Immunoblot for MDA5 proteins in lysates from E. Data show means ± SD from four (A and E), three (C), and five experiments (D). Data in B and F are examples from experiments shown in A and C–E, respectively. 293T cells lack endogenous MDA5 expression (not depicted). Equivalent lysates from ∼30,000 cells were run across lanes. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, by one-way ANOVA.