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. 2017 Mar 28;7:45375. doi: 10.1038/srep45375

Figure 4. Nedd4 regulates the replication of JEV and influences viral yields.

Figure 4

(A) Cells transfected with Nedd4 siRNA (50 nM) or the NT siRNA (50 nM) were infected with JEV (MOI = 1.0), and viral RNA levels were determined by RT-qPCR at 2 h, 12 h, and 24 h p.i. (B) JEV NS3 expression was evaluated by Western blot in Nedd4-depleted cells. GAPDH served as a control for equal sample loading. The level of NS3 was quantitated by densitometric analysis using ImageJ software and normalized to GAPDH. (C) The virus titres of the culture supernatants from Nedd4-depleted cells were determined by plaque forming unit assays. (D) Cells transfected with a plasmid expressing human Nedd4 or with an empty vector were infected with JEV (MOI = 1.0). Nedd4 and JEV NS3 expression was evaluated by Western blot. (E) The virus titres of the culture supernatants from Nedd4-overexpressed cells were determined by plaque forming unit assays. (F) Cells were infected with JEV (MOI = 1.0) for 1 h. The cells were then washed with 10% FBS DMEM and treated with CysA (5 μg/ml) in 10% FBS DMEM for 24 h. Nedd4 and JEV NS3 expression was evaluated by Western blot. The experiments were performed independently in triplicate. The data are shown as the mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001.