Mouse anti-FLAG (M2) and anti–β-actin Abs were from Sigma-Aldrich (St. Louis, MO), whereas mouse anti-Myc (9E10) and anti-V5 Abs were from Santa Cruz Biotechnology (Dallas, TX) and Life Technologies, respectively. Rabbit anti-MDA5 (AT113) antiserum was from Enzo Life Sciences (Farmingdale, NY), and rabbit anti-PACT (ab31967) Abs were from Abcam (Cambridge, U.K.). HRP-conjugated anti-mouse and anti-rabbit IgG secondary Abs were from GE Healthcare Bio-Sciences (Pittsburgh, PA). Using these Abs, poly(I:C) pull-down, coimmunoprecipitation, and Western blotting were performed as previously described (20 (link), 27 (link)). Nondenaturing native PAGE for IRF3 dimerization was performed accordingly (28 (link)), whereas that for MDA5 oligomerization was modified to a 5% gel.
Confocal microscopy was performed using a Carl Zeiss LSM710 microscope, as described (25 (link), 29 (link)). JEG-3 cells were transfected with expression plasmids for 48 h and then induced with poly(I:C) for 3 h. The cells were fixed with 4% paraformaldehyde, permeabilized with methanol-acetone (1:1), and blocked with 3% BSA. FLAG-MDA5 and V5-PACT were detected with rabbit anti-V5 (V8137) and mouse anti-FLAG (F1804; both from Sigma-Aldrich) Abs, respectively, and nuclei were visualized with DAPI.
Confocal microscopy was performed using a Carl Zeiss LSM710 microscope, as described (25 (link), 29 (link)). JEG-3 cells were transfected with expression plasmids for 48 h and then induced with poly(I:C) for 3 h. The cells were fixed with 4% paraformaldehyde, permeabilized with methanol-acetone (1:1), and blocked with 3% BSA. FLAG-MDA5 and V5-PACT were detected with rabbit anti-V5 (V8137) and mouse anti-FLAG (F1804; both from Sigma-Aldrich) Abs, respectively, and nuclei were visualized with DAPI.