English: (A and B) The reporter assays using the ISG54 ISRE-luciferase construct. 293T cells in 12-well plates were cotransfected with 0.5 µg of the ISG54 ISRE-luciferase reporter construct and plasmids expressing HA-IE1, flag-SUMO-1, HA-PIAS1, and myc-IE2(346–542) as indicated. The total amount of plasmid was adjusted with empty vectors. At 24 h, cells were untreated or treated with IFNβ (1,000 units/ml) for 8 h, and luciferase reporter assays were performed. The results shown are the mean values and standard errors of three independent experiments. Statistical significance between samples was determined using Student's t-test (values of *P<0.0005). The expression levels of IE1, IE2, and β-actin proteins in cell lysates were determined by immunoblotting with specific antibodies. (C) A hypothetical model showing that expression of IE2 and its SUMOylation regulates the PIAS1-mediated IE1 SUMOylation, enhancing IE1 activity to downregulate type I IFN-stimulated gene (ISG) expression. ISRE, interferon stimulated response element.