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. 2019 Jul 30;93(16):e00336-19. doi: 10.1128/JVI.00336-19

FIG 7.

FIG 7

Comparative analysis of MAPK/ERK pathway for NS3 proteins from different orbiviruses. (A and C) HEK-293T cells were transfected with expression vectors encoding GST alone or fused to BTV8-NS3, BTV1-NS3, BTV27-NS3, EHDV-NS3, AHSV-NS3, or EEV-NS3 as indicated and tested for interaction with endogenous BRAF. Total cell lysates were prepared at 48 h posttransfection (cell lysate; middle panel), and copurifications of endogenous BRAF were assayed by pulldown using glutathione-Sepharose beads (pulldown; upper panel). Sizes are shown in kilodaltons (kDa). (B and D) As described in Fig. 1, HEK-293T cells were transfected with pFA2-Elk1, pGal4-UAS-Luc, and pRL-CMV. In addition to these three plasmids, cells were cotransfected with an expression vector encoding 3×FLAG-tagged BTV8-NS3, BTV1-NS3, BTV27-NS3, EHDV-NS3, AHSV-NS3, EEV-NS3, or the corresponding empty vector pCI-neo-3×FLAG, as indicated. At 12 h after transfection, the cells were serum starved for 24 h, and the relative luciferase activity was then determined. The experiment was performed in triplicate, and data represent means ± the SD. ***, Differences observed between BTV8-NS3, BTV1-NS3, or BTV27-NS3 and the corresponding control vector pCI-neo-3×FLAG were statistically significant (P < 0.0005); **, differences observed between BTV-NS3 and the corresponding control vector pCI-neo-3×FLAG were statistically significant (P < 0.005); ns, nonsignificant differences between EHDV-NS3, AHSV-NS3, or EEV-NS3 and the corresponding control vector pCI-neo-3×FLAG.