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. 2007 Aug 8;81(20):11226–11235. doi: 10.1128/JVI.00431-07

FIG. 3.

FIG. 3.

BiFC analysis of EIAV and HIV-1 Gag assembly in human cells. (A) Visualization of Gag assembly by BiFC assay. 293T (panels a to d) and HeLa (panels e to h) cells grown on glass coverslips were transfected with Rev-independent EIAV-Gag-VN/Gag-VC (panels a and e), Rev-dependent EIAV-Gag-VN/Gag-VC (panels b and f), Rev-independent HIV-Gag-VN/Gag-VC (panels c and g), and Rev-dependent HIV-Gag-VN/Gag-VC (panels d and h) plasmid pairs using Lipofectamine 2000. At 16 h posttransfection, cells were fixed and imaged with a Leica TCS-SL confocal microscope. Bar, 10 μm. (B) Quantitative analysis by flow cytometry of BiFC signals in transient transfected HeLa cells described in panel A. At 16 h posttransfection, permeabilized cells were stained with mouse HA antibody and Cy5-conjugated goat anti-mouse IgG. A minimum of 30,000 gated live events were acquired and analyzed by both yellow and HA-Cy5 fluorescence. The percentage of HA and BiFC double-positive cells in total HA-positive cells was calculated and labeled below each quadrant plot.