Fig. 5: UBA3 activates ISG production.

a Western blot detection of UBA3 and GAPDH in WT and UBA3 KO (sgRNA1, sgRNA2) HEK293 cells. Experiments were repeated two times. b WT, UBA3 KO (sgRNA1, sgRNA2), PFDN4 KO, PFDN4 and UBA3 double KO (sgRNA1, sgRNA2) HEK293 cells were stimulated with IFN-β (500 U/ml) for 24 h. MX1 mRNA level was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Dunnett’s multiple comparisons test). c WT, UBA3 KO (sgRNA1, sgRNA2), PFDN4 KO, and PFDN4 and UBA3 double KO (sgRNA1, sgRNA2) HEK293 cells were stimulated with IFN-β (500 U/ml) for 24 h. Cell lysates were analyzed by western blot. d WT, UBA3 KO HEK293 cells were infected with or without RV (RRV strain, MOI of 5) for 24 h. MX1 mRNA level was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (two-way ANOVA with Tukey’s multiple comparisons test). e WT and UBA3 KO HEK293 cells were treated with or without Ruxo (10 μM) and IFN-β (500U/ml) for 24 h, and then infected with UK (MOI = 5) for 72 h. Virus titers were determined by an FFU assay. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Tukey’s multiple comparisons test). f WT and UBA3 KO HEK293 cells were induced with IFN-β (500 U/ml) for 24 h. IRF9 mRNA level was measured by RT-qPCR. Data represents the average of three experiments; error bars indicate SEM (one-way ANOVA with Tukey’s multiple comparisons test). g WT and UBA3 KO HEK293 cells were treated with DMSO or MLN4924 (1 μM) for 24 h, then induced with IFN-β (500 U/ml) for 24 h. Cell lysates were analyzed by western blot.