Figure 6.

Ctenopharyngodon idella melanoma differentiation-associated gene 5 (CiMDA5) and C. idella retinoic acid-inducible gene I (CiRIG-I) affect the phosphorylation levels of grass carp IFN regulatory factor 3 (CiIRF3) and CiIRF7 under grass carp reovirus (GCRV) infection or polyinosinic:polycytidylic acid [poly(I:C)] stimulation. (A,C) MDA5+, retinoic acid-inducible gene I (RIG-I)+, and EGFP+ cells were seeded into 6-well plates, and then either treated with phosphate-buffered saline, infected with GCRV, or stimulated with poly(I:C) for 24 h. Whole-cell lysates (WCL) were prepared for Western blot analysis. “p” in the front of IRF3\7 indicates the phosphorylation. (B,D) C. idella kidney cells were seeded in 10-cm plates, and then infected with GCRV for 24 h. WCL were subjected to immunoprecipitation (IP) with 1 μg of corresponding antiphosphorylation antibodies, followed by Western blotting analysis. (E,F) MDA5+, RIG-I+, and EGFP+ cells were seeded into 10-cm plates, and then either infected with GCRV or stimulated with poly(I:C). Twenty-four hours later, IP assays were performed with antiphosphoserine antibody or antiphosphothreonine antibody. The histograms exhibit the relative expression levels, which are quantified using ImageJ software. All the immunoblots were performed using anti-IRF3 or anti-IRF7 anti-rabbit polyclonal antibody.