(A) HEK293 cells were transfected with MDA5 expression plasmid, alone or together with PACT expression plasmid, and harvested for a poly (I:C) pull-down experiment. The bound fraction of poly(I:C)-conjugated beads was evaluated for MDA5 recruitment at 15 and 30 min after lysate incubation. (B) HEK293 cells were transfected with MDA5 and PACT expression plasmids, alone or in combination for 30 h, and then subjected to mock treatment or poly (I:C) induction. Coimmunoprecipitation was carried out with anti-FLAG or anti-V5 Ab. (C) JEG-3 cells were transfected with expression plasmids of MDA5 and PACT and subjected to mock treatment or poly(I: C) induction. Subcellular localization of PACT (red) and MDA5 (green) was visualized by confocal microscopy. Nuclear morphology (blue) was revealed by DAPI staining. Relative colocalization (yellow) is shown in the merged image. Condensation of the merged signal is indicated by the arrowhead. Scale bars, 20 μm.