MAVS Y9F impairs MAVS-mediated innate immune signaling.
(A) Expression of wild-type (WT) and mutant MAVS proteins was assessed by Western-blotting in HEK293T cells. (B–C) HEK293T cells (B) or MCF-7 cells (C) were transfected with expression vector encoding Flag-MAVS WT or Flag-MAVS mutants together with IFN-β-LUC or NF-κB-LUC. The LUC activity was measured 24 h later and normalized for transfection efficiency. Results are expressed as fold-increases of luciferase levels relatively to Flag-vector transfected cells. (D) HEK293T cells were transfected with a vector encoding Flag-MAVS or Flag-MAVS mutants and endogenous IFN-β mRNA expression was analyzed by qRTPCR. Results were expressed as fold-increase of IFN-β mRNA level normalized with β-actin level and relatively to Y9F MAVS-transfected cells. (E) Using the supernatants collected from the samples shown in panel B, IL6 release was measured by ELISA. From B to E, Data are mean of three independent experiments ± SEM done in triplicate.(F) HEK293T cells were transfected with expression vector encoding Flag-MAVS WT (WT) or Flag-MAVS Y9F mutant (Y9F). Whole cell lysates were analyzed by immunoblotting with anti-IRF3, anti-p-IRF3 or anti-Flag antibody, anti-Tubulin was used as equal loading control. (G) EMSA was performed using 32P-labeled consensus NF-κB probe and nuclear proteins extracted from HEK293T cells transfected with Flag-MAVS WT (WT) or Flag-MAVS Y9F mutant (Y9F).
