Fig. 4.

miR-33/33* inhibit the formation of functional MAVS aggregates to suppress RIG-I signaling. a and b Immunoblot analysis of phosphorylated (p-) or total proteins in lysates of macrophages (MΦ) a or HeLa cells b transfected with nc, miR-33, or miR-33* or inc, miR-33 in, or miR-33* in followed by infection for the indicated hours with VSV (MOI = 1). c IFN-β promoter luciferase reporter activity (IFN-β-Luc) in HEK293T cells co-transfected with nc, miR-33, or miR-33* or inc, miR-33 in, or miR-33* in, together with empty vector or an expression plasmid for the RIG-I amino-terminal ‘2CARD’ domain (RIG-IN), MAVS, TBK1, or IRF3-5D. d SDD-AGE analysis and SDS-PAGE analysis of the aggregation of MAVS in macrophages transfected as in a followed by infection for the indicated hours with VSV (MOI = 1). e SDD-AGE analysis and SDS-PAGE analysis of the aggregation of MAVS in HEK293T cells transfected with nc, miR-33 plus miR-33* (miR-33/33*), inc, or miR-33 in plus miR-33* in (miR-33/33* in) along with Flag-MAVS. f Immunofluorescence with confocal microscopy of HeLa cells transfected as in a along with the Mito-YFP plasmid and Flag-MAVS. Cellular nuclei are stained with the DNA-binding dye DAPI; Flag-MAVS is visualized using primary antibodies to MAVS followed by a secondary antibody expressing DyLight 594; enlarged region, enlargement of the area outlined on the left; surface rendered column, 3D surface rendering process of left column using Imaris 9.0. Scale bar, 2 µm. *p < 0.05 (one-way ANOVA c). Data are presented as the mean ± s.e.m. and are representative of three independent experiments.