FIG 3.

Stimulation of ERK1/2 and eIF4E phosphorylations by BTV infection or BTV-NS3 expression. HEK-293T cells were transfected with an expression vector encoding 3×FLAG-tagged BTV-NS3 or the corresponding empty vector pCI-neo-3×FLAG (B). After 18 h, the cells were serum starved and, where indicated, infected with WT BTV (MOI = 0.01) (A). After 12 h, the cells were stimulated with 400 ng/ml of EGF. Phosphorylations of ERK1/2 and eIF4E were measured at 10 min, 30 min, 2 h, 6 h, and 24 h after EGF stimulation (A and B). BTV infection was confirmed by anti-NS3 immunoblotting (A, lower panel), and the expression of 3×FLAG-tagged BTV-NS3 in transfected cells was detected by anti-3×FLAG immunoblotting (B, lower panel). Densitometric analysis of the gels were performed for p-ERK, ERK1/2, peIF4E, and eIF4E, and the graphs represent the ratio of phosphorylated proteins to total proteins. Data presented are representative of at least three independent experiments. Sizes are shown in kilodaltons (kDa).