Figure 5.

Dimerization-defective Nef mutants interact with Itk but fail to induce kinase activation. A, Itk was expressed either alone or together with WT Nef or dimerization-defective mutants as BiFC pairs in 293T cells. The cells were fixed and stained for confocal microscopy with anti-pTyr antibodies as a measure of kinase activity (red) and anti-V5 antibodies to verify Itk protein expression (blue). Nef interaction with Itk is observed as Venus fluorescence (BiFC; green). B, single-cell image analysis was performed as per the legend to Fig. 1. The fluorescence intensity ratio (pTyr:Itk expression) for each cell is shown as a horizontal bar, with the median value indicated by the red bar. Student's t tests were performed on the groups indicated by horizontal lines above the plot; p < 0.0001 in each case.