NOG1 impairs the DNA binding ability of IRF3.

HEK-293T cells transfected with increasing Flag-NOG1 expression plasmid (0, 1, and 3 μg) (A), and WT and NOG1+/- HEK-293T cells (B and C) were infected with SeV for 12 h. Chromatin was immunoprecipitated with an anti-IRF3 antibody. The impact of NOG1 on IRF3 binding onto IFN-β and ISG56 promoter was analyzed by quantitative ChIP assay. PCR products were analyzed by nucleic acid electrophoresis. The abundance of the immunoprecipitated DNA was normalized to the input DNA levels using ImageJ Software. (D) The effect of NOG1 on IRF3 binding onto ISG54 promoter. HEK-293T cells cotransfected with HA-IRF3 and Flag-NOG1 were infected with SeV for 12 h. Biotinylated and unbiotinylated promoters were mixed with the cell lysates. The streptavidin beads were then added and incubated, followed by washing with PBS. Western blotting was performed to detect IRF3 and NOG1.