Fig. 4

NS1 with 188-Val antagonizes IFN-β production through targeting TBK1. a, b HEK-293T cells were co-transfected with IRF3-expressing plasmid, RIG-I(2CARD)-encoding plasmid as a stimulator, FSS WT NS1-expressing plasmid or FSS NS1 A188V-expressing plasmid (a), PRV WT NS1-expressing plasmid or PRV NS1 V188A-expressing plasmid (b). At 24 h post-transfection, cells were harvested and lysed, and cell extracts were subjected to western blotting with indicated antibodies. c, d Plasmids expressing TBK1 (c) or IKKε (d), together with FSS13025 WT, FSS13025 A188V, PRVABC-59 WT, or PRVABC-59 V188A expression plasmids were co-transfected into HEK-293T cells. Cells were harvested at 24 h post-transfection and analyzed by western blotting for pTBK1 (anti-pTBK1 S172), total TBK1 (anti-TBK1), pIKKε (anti-pIKKε S172), total IKKε (anti-IKKε), GAPDH (anti-GAPDH), and NS1 protein (anti-HA). e, f Co-immunoprecipitation for detection of NS1 and TBK1 interaction. HEK-293T cells were transfected with TBK1-encoding plasmid and HA-tagged FSS NS1, FSS NS1 A188V, PRV NS1, or PRV NS1 V188A expression plasmid. At 24 h post-transfection, cells were harvested and whole-cell extracts (WCE) were used for immunoprecipitation using anti-HA beads (e) or anti-TBK1 antibody (f), followed by immunoblot with indicated antibodies. The original uncropped blots can be found in Supplementary Fig. 7