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. 2017 Jan 31;91(4):e02124-16. doi: 10.1128/JVI.02124-16

FIG 5.

FIG 5

Effects of mutations in NS4A on the subcellular localization of the NS3 and NS4A proteins. (A, B) V5-tagged NS3 and/or HA-tagged NS4A expression plasmids were transfected into Huh-7.5.1 cells. On the following day, the cells were fixed and permeabilized. V5-tagged proteins were visualized by staining with anti-V5 antibody, and HA-tagged proteins were visualized by staining with anti-HA antibody. (C) Ratios of the NS3 distribution (cytoplasmic/nuclear). The average intensities of the NS3 protein in the cytoplasmic and nuclear areas were quantified with ImageJ software, and the ratios were calculated. (D) BiFC assay of NS3 and NS4A. The NS3 expression plasmid with the mKG-N fragment (phmKGN/NS3-JFH1, phmKGN/NS3-J6, or phmKGN/NS3-J6/N3H-JFH1) and the NS4A expression plasmid with the mKG-C fragment (phmKGC/4A-JFH1, phmKGC/4A-J6, phmKGC/4A-J6/W1664S, phmKGC/4A-J6/A1676T, phmKGC/4A-J6/A1680E, or phmKGC/4A-J6/T1681S) were cotransfected into Huh-7.5.1 cells. On the following day, the cells were fixed and examined using a fluorescence microscope. Positive control, mKG-fused p50 and p65 partial domains from NF-κB complex; negative control, empty vectors.