Fig. 4.

TULV C42 inhibits TBK1-directed ISRE and IFN-β activation. (A) HEK293 cells were transfected with an ISRE-driven luciferase reporter construct in the presence or absence of an activating TBK1 expression vector. Cells were cotransfected with plasmids expressing PHV, TULV Gn-T-C42, or an empty vector control (pBIND expressing the Gal4 tag) to maintain DNA transfection levels. (B) HEK293 cells were transfected with an IFN-β-driven luciferase reporter construct and with or without an activating TBK1 expression vector. Cells were cotransfected with plasmids expressing PHV Gn-T-C42, increasing amounts of TULV Gn-T-C42, or an empty vector control (pBIND) to maintain DNA transfection levels. Two days posttransfection, luciferase activity was measured as described in the legends of previous figures, and values are reported as the fold increase compared to controls lacking TBK1. Assays were performed in triplicate with similar results in at least two separate experiments. Western blot (WB) analysis showing equal expression of TULV Gn-T-C42 and PHV Gn-T C42 (B) was performed. Cells were lysed at 48 h posttransfection, and equal amounts were loaded onto a 12% SDS-PAGE gel. Proteins were detected by Western blotting using anti-Gal4 (Santa Cruz Biotechnology) and anti-mouse HRP-conjugated secondary antibody. Blots were stripped and reprobed with anti-β-actin (Sigma).