Fig. 4

UBE2J1 negatively regulates IRF3-mediated IFNβ signaling. a IFNβ mRNA expression in scramble shRNA (N.C.) or UBE2J1 shRNA transfected HEK293T cells after DENV infection. b Protein levels of IFNβ in scramble shRNA (N.C.) or UBE2J1 shRNA transfected HEK293T cells after DENV infection. c HEK293T cells were co-transfected with the IFNβ luciferase reporter, RIG-I-N (or vector), pRL-TK and UBE2J1 (or N.C.) shRNA plasmids. At 24 h after transfection, luciferase reporter assay was used to analysis of IFNβ promoter activity. d HEK293T cells were transfected with vector or UBE2J1 overexpression plasmids and infected with DENV. At 24 h or 48 h post infection, IFNβ mRNA expression was analyzed by qRT-PCR. e DENV induced IFNβ protein in UBE2J1 overexpressing or control HEK293T cells as quantified using ELISA. f Effect of overexpression of UBE2J1 on IFNβ reporter activation directed by RIG-I-N, MAVS, TRAF3, TBK1, IKKε, or IRF3-5D. Western blots were performed to confirm the overexpression of indicated proteins. g-i UBE2J1 suppressed DENV induced ISRE-promoter activity and ISG mRNA production. HEK293T cells were transfected with UBE2J1 (or N.C.) shRNA and then treated with DENV (or Mock) infection. At 24 h post infection, ISRE promoter activities were analyzed by luciferase assay (g) and ISG15 (h) and MxA (i) mRNA expression were measured by qRT-PCR. j Overexpression of UBE2J1 downregulated RIG-I-N, or IRF3-5D induced IRF3 promoter (IRF3-Luc) activity. k Western blots were used to confirm the overexpression of indicated proteins shown in data J. Results were expressed as the mean + SD. *p < 0.05 and ** p < 0.01 (t-test). Representative results from at least 3 independent experiments