FIGURE 10.

Interaction of Nef Arg-105 with Hck SH3 Glu-93 is required for complex formation in vivo. Interaction of Nef with truncated and full-length Hck proteins was assessed using a cell-based BiFC assay. Human 293T cells were transfected with expression plasmids for fusion proteins of Nef with an N-terminal fragment of the YFP variant, Venus, and either the N-terminal region of Hck including the SH3 domain or full-length Hck fused to the complementary C-terminal Venus fragment. Parallel experiments were conducted with wild-type (WT) and SH3 domain mutants in which Glu-93 was replaced with alanine (E93A). Eighteen hours after transfection cells were fixed and stained with antibodies to the Hck N-terminal region and Nef. Three-color confocal images were recorded for BiFC (green), which is indicative of Nef·Hck interaction as well as Hck and Nef protein expression. Mean pixel intensities of the BiFC and immunofluorescent (IF) signals were determined for at least 50 cells per condition using ImageJ. BiFC:immunofluorescent signal ratios were calculated and are presented in the bar graph as the mean ratio ± S.E.