Figure 7.

The same and differential effects of Ctenopharyngodon idella melanoma differentiation-associated gene 5 (CiMDA5) and C. idella retinoic acid-inducible gene I (CiRIG-I) on the dimerization of grass carp IFN regulatory factor 3 (CiIRF3) and CiIRF7. (A) Applicability test for anti-HA, -Flag, -myc antibodies. C. idella kidney (CIK) and EGFP+ cells seeded in 6-well plates were harvested for Western blot assay. Anti-HA, -Flag, -myc antibodies were used. Dotted boxes show the areas of corresponding target protein bands. (B–D) Grass carp reovirus (GCRV) infection facilitates the homo- and heterodimerization of IRF3 and IRF7. CIK cells seeded in 10-cm dishes were co-transfected with 4 μg pIRF3-Flag and 4μg pIRF3-myc (B), pIRF3-Flag and pIRF7-myc (C), and pIRF7-Flag and pIRF7-myc (D). Twenty-four hours later, the transfected cells were infected with GCRV. At 24 h postinfection, cells were harvested for Co-IP assay with anti-myc antibody and subsequently immunoblotted with the indicated antibodies. (E–G) Effect of CiMDA5 and CiRIG-I on the dimerization of CiIRF3 and CiIRF7. CIK cells seeded in 10-cm dishes were co-transfected with 2.6 μg of pMDA5-HA (or pRIG-I-HA, or pdCMV), 2.6 μg of pIRF3-Flag and 2.6 μg of pIRF3-myc (E), pIRF3-Flag and pIRF7-myc (F), pIRF7-Flag and pIRF7-myc (G). The subsequent experiments were carried out as described above.