Fig 4.

NS4A forms AH-dependent oligomers. (A) Immunoprecipitation of wild-type NS4A and AH mutants. HEK293T cells were cotransfected with the indicated combinations. The cells were lysed 24 h posttransfection and subjected to immunoprecipitation using anti-FLAG beads followed by Western blotting with an anti-HA antibody. Direct Western blotting of the cell lysates with anti-FLAG and anti-HA antibodies are shown in the lower panels. Molecular mass markers are indicated on the left. (B) NS4A CFP and YFP fusion constructs and AH mutants thereof were transfected into U2OS cells as indicated. FRET was determined using acceptor photobleaching. Fluorescence of donor and acceptor was measured as a function of time during acceptor photobleaching. Intensities within the labeled (white square) region of interest were analyzed and are plotted in the graphs. Bar = 5 μm. Asterisks denote statistical significance in an up-paired Student t test. **, P < 0.05; ***, P < 0.0.005.