Figure 3. Ub2D3 and Ube2N are required for RIG-I and MAVS activation on virus infection in HEK293T cells.

(a,b) The cell-free assay containing vRNA and purified components (E1, Ub, RIG-I and Riplet) was performed as described in Fig. 2b, except that S100 fraction was substituted with purified E2 recombinant proteins as indicated. Each E2 protein was analysed at three different concentrations. Lane 13 did not contain E2 proteins (−E2). (c) HEK293T cells (wild-type and knockout lines) were transfected with or without si-Ube2D3 RNAs. Seventy two hours after transfection, the cells were infected with VSV. Twelve hours post virus infection, the cells were collected for measuring IFN induction by qPCR. *P<0.05 and ***P<0.001. (d,e) Cells were treated as described in c. P5 fractions were isolated to examine MAVS aggregation (d), and whole cell lysate were used to analyse knockdown or knockout effect in genes as indicated (e). The original full blot for (e) can be found in Supplementary Fig. 8d. (f) si-Ube2D3 RNAs were transfected into HEK293T (Ube2N^−/−) cells. Seventy two hours after siRNA transfection, the cells were further transfected with pcDNA-flag-Ube2D3 or pcDNA-flag-Ube2N as indicated. Twenty four hours after transfection of pcDNA plasmids, the cells were infected with VSV for another twelve hours. The cells were then collected for analysis of MAVS aggregation. See also Supplementary Fig. 3f.