FIG. 3.

Quantification of inhibition of STAT1 phosphorylation by flavivirus NS5 and 2KNS4B proteins. (A) Western blot demonstrating the expression of NS5 and 2KNS4B proteins, each in frame with a C-terminal V5 epitope tag, in Vero cells. (B) Flow cytometry analysis to determine the percent inhibition of pY-STAT1 in Vero cells expressing flavivirus nonstructural proteins. Vero cells were fixed, permeabilized, and stained using antibodies that recognize pY(701)-STAT1 and V5. The examples shown are cells transfected with an empty plasmid and either untreated or treated with IFN-β (top panels) or cells expressing LGTV NS5 or WNV-NY99 NS5 and treated with IFN-β (lower panels). (C) Quantification of pY-STAT1 inhibition by each protein analyzed by flow cytometry. V5-positive cells were gated and examined for pY-STAT1. The percent pY-STAT1 inhibition is the percentage of V5-positive cells (contained in quadrant 2 [Q2] and Q4 in B) that were pY-STAT1 negative (in Q4). The average level of V5-negative cells that are also pY-STAT1 negative in IFN-β-treated cultures (cells present in Q3) is illustrated by the dotted line as an indication of the background levels in this assay. Error bars indicate SEM from four independent experiments; asterisks indicate significant differences (P < 0.05, by ANOVA followed by Tukey's test).