IRS4 diminished the inhibitory effects of USP18 on Jak/STAT signaling pathway (A-C). 293T cells (A) and Huh7.5.1 cells (B) were transfected with single empty plasmid (M, mock control), Flag-USP18 and Myc-IRS4, or co-transfected with USP18 and IRS4. The cells were treated with 100IU/mL IFN-α for 30 mins at 48 hours after transfection. Protein lysates were harvested, separated by SDS-PAGE, and probed for STAT1-phospho701 and total STAT1 expression. The ratio of p-STAT1 to total STAT1 from 3 independent experiments was quantified (bottom). (C) USP18 expression Flag-tagged plasmid (M, mock control), IRS4 expression Myc-tagged plasmid were co-transfected together with plasmids pISRE-luc and pRL-TK into Huh7.5.1 cells as described in Materials and Methods. 24 hours later, cells were treated with 100IU/mL IFN-α for 24 hours before the cells were lysed for dual luciferase reporter gene assay for ISRE activity. Error bars indicated mean±SD, *P<0.05, **P<0.01, ***P<0.001.