FIG 3.

NS5A S235 phosphorylation was a prerequisite to S238 phosphorylation. Immunoblots for NS5A phosphorylation at S222 (A), S235 (B), and S238 (C) in the HEK293T cells. The HEK293T cells were transfected with various NS3-NS5A expression constructs: the wild types (WT) and single-, double-, or triple-alanine mutations at S222, S235, or S238 for 2 days before the cell lysates were collected for immunoblotting. ß-Actin staining served as a loading control. (D) A bar diagram summarizing the immunoblotting results from three experiments. Values are means ± SE (n = 3). The asterisk indicates significance: P value of <0.05 by Student's t test. (E and F) Representative immunoblot images (E) and summary (F) for NS5A phosphorylation at S235 and S238 in the HEK293T cells transfected with an S235D mutant NS3-NS5A expression construct.