Fig. 3.

pS273R suppresses IRF3 phosphorylation and dimerization. A HEK293T cells were transfected with Flag-cGAS (100 ​ng) and Flag-STING (320 ​ng) plasmids, and different doses of HA-S273R (0, 0.5, 1, and 2 ​μg) or empty vector. Cells were harvested at 24 hpt. Western blotting was used to examine cGAS, STING, TBK1, p-TBK1, IRF3, p-IRF3, pS273R, and β-actin. B 3D4/21 ​cells were transfected with empty vector or Myc-S273R (1 ​μg) for 24 ​h and then stimulated with poly(dA:dT) (1 ​μg/mL) for 12 ​h. The cells were harvested for Western blotting analysis. C HA-S273R plasmid (500 ​ng or 1000 ​ng) was co-transfected with Flag-TBK1 (200 ​ng) into HEK293T cells. After 24 ​h, cells were harvested for Western blotting analysis. D HEK293T cells were transfected with Myc-S273R for 12 ​h and then infected with VSV (MOI ​= ​1) for 12 ​h. Cells were lysed for Western blotting analysis to determine the levels of p-TBK1, TBK1, p-IRF3, IRF3, pS273R, and β-actin. E HEK293T cells were transfected with Flag-IRF3, HA-IRF3, HA-TBK1, and Myc-S273R (1 ​μg or 2 ​μg). At 24 hpt, cells were harvested for co-immunoprecipitation analysis. F HEK293T cells were transfected with pRK5-HA (1 ​μg) or HA-S273R for 12 ​h and then infected with VSV (MOI ​= ​1) for 12 ​h. Whole cell extracts were subjected to native PAGE. Dimers or monomers of IRF3, pS273R, and β-actin were detected with the respective antibodies.