The GTP binding domain of NOG1 is responsible for the inhibition of IFN-β.

(A) HEK-293T cells were transfected with 0.1 μg of IFN-β-Luc and 0.01 μg of pRL-TK plasmid along with 0.1 μg of Flag-NOG1- or Flag-NOG1-mutants-expressing plasmids (left). HEK-293T cells were transfected with 0.5 μg of Flag-NOG1- or Flag-NOG1-mutants-expressing plasmids (right). At 24 hpt, the cells were infected with SeV for 12 h. The promoter activation of IFN-β was determined by the dual-specific luciferase assay kit. The expression of IFN-β protein in the supernatant was detected by ELISA kit. The protein expression of NOG1 and its mutants was detected by Western blotting. (B) HEK-293T cells were transfected with 3 μg of Flag-NOG1- or Flag-NOG1-mutants-expressing plasmids. At 24 hpt, the cells were infected with SeV for 12 h. The cell lysates were immunoprecipitated with an anti-Flag antibody and subjected to Western blotting. (C) HEK-293T cells transfected with 3 μg of Flag-NOG1 or Flag-NOG1 GKS-AAA expression plasmid were infected with SeV for 12 h. The cells were collected and analyzed by quantitative ChIP assay. The abundance of PCR production was normalized to the input DNA levels using ImageJ Software.