Figure 3.

Membrane recruitment of Btk and Itk requires myristoylation of HIV-1 Nef. A, Itk or Btk were expressed either alone or in combination with WT or myristoylation-defective Nef (G2A) in 293T cells. The cells were stained with antibodies to the V5 tag present in each kinase protein, and imaged via confocal microscopy. Representative images are shown. B, using the Plot Profile tool from ImageJ, a line was drawn across the cytoplasm of representative cells from each condition, excluding the nucleus. The change in fluorescence intensity along this line was then plotted as relative pixel density as a function of distance. Very similar results were observed with at least 10 cells from each condition, and a representative example is shown. Strong membrane localization of both Itk and Btk was observed in the presence of WT Nef and is reflected by the prominent peaks indicated by the red arrows.