Figure 6.

The identification of IKKε as the cellular target by ASFV pS273R. (a–e) HEK293T cells were co-transfected with 20 ng TBK1 expression plasmids and 10 ng 3×FLAG-pCMV-S273R (a), with 20 ng IKKε expression plasmid and increased amounts of pS273R (10 ng or 20 ng) (b), with 20 ng IRF3-5D expression plasmid and 10 ng pS273R (b), with 20 ng IKKβ plasmid and 10 ng pS273R (d), with 20ng p65 plasmid and 10 ng pS273R (e), plus 10 ng ISRE-luc, IFNβ-luc or NF-κB-luc and 0.2 ng pRL-TK plasmids, which were normalized to 50 ng/well by vector 3×FLAG-pCMV. After 24 h, the luciferase activities were detected using Double-Luciferase Reporter Assay. (f,g) pS273R plasmid (400 ng or 800 ng) were co-transfected with 400 ng IKKε into HEK293T cells. After 24 h, the cells were harvested and analyzed by RT-qPCR (f) and Western blotting (g). (h-j) the IKKε plasmid (500 ng) were co-transfected with 500 ng pS273R plasmid or empty vector into 293T cells for 24 h, then the transfected 293T cells were infected with 0.01 MOI or .1 MOI HSV-1-GFP for 36 h, the GFP signals were observed by fluorescence microscopy (H). the infected cells were harvested to measure the HSV-1 gene expression by RT-qPCR (i). the viral titer in the supernatant from HSV-1 infected 293T cells was measured by TCID50 assay (j).