Figure 2.

Nef induces constitutive activation of Btk at the cell membrane. A, Btk was expressed either alone or together with WT or myristoylation-defective Nef (G2A) as BiFC pairs in the absence or presence of the Btk inhibitor, ibrutinib (1 μm) 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 Btk protein expression (blue). Nef interaction with Btk is observed as fluorescence complementation of the YFP variant, Venus (BiFC; green). B, single-cell image analysis was performed as per the legend to Fig. 1. The fluorescence intensity ratio (pTyr:Btk expression) for each cell is presented as a horizontal bar, with each 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 (***).