Figure 3
Both TRAF3 and TRAF6 play a critical role in SeV-induced signaling in HEK293T cells.
(A) 293T cell pools with stable knockdown of TRAF3, TRAF6 and VISA were generated by shRNA. Knockdown efficiency was analyzed by western blot with anti-TRAF3, TRAF6 and VISA antibodies. (B) SeV-induced IFN-β production was reduced when endogenous TRAF3, TRAF6, or VISA was stably knocked down. Indicated cells (5 × 105) were infected with SeV and IFN-β levels were estimated by Q-PCR (B, left) and bioassay (B, right) at the indicated times. (C) IRF3 dimer and p-IRF3 were attenuated in 293T-shTRAF3, 293T-shTRAF6 and 293T-shVISA cells infected with SeV. Indicated cells (5 × 105) were infected with SeV for the indicated times and lysates were analyzed by western blot with anti-p-IRF3 and anti-GADPH or by native western blot with anti-IRF3. (D) TRAF3 and TRAF6 knockout HEK293T cell lines were generated by CRISPER/CAS9. Knockout cells were analyzed by western blot with anti-TRAF3, TRAF6 and VISA antibodies. (E) p-IRF3 was attenuated in TRAF3-knockout (KO) 293T, TRAF6-KO 293T and VISA-KO 293T cells infected with SeV. Indicated knockout cells (5 × 105) were infected with SeV and lysates were analyzed by western blot with anti-p-IRF3 antibody. (F) SeV-induced IFN-β was reduced when endogenous TRAF3, TRAF6 and VISA were knocked out. Q-RT-PCR (left) and IFN-β bioassay (right) were performed as in (B). Each graph represents the mean ± SD of three independent experiments done in triplicate. ***indicates P < 0.001; **indicates P < 0.01; ns (not significant) indicates P > 0.05.
