Effect of Unc119 G22V on Lck localization. (A) Peripheral blood CD4 T cells from P1 and a healthy control subject (C) were immunostained for Lck (red) and inspected with a fluorescent microscope (magnification ×1000). Z-stacks of images were collected and subjected to three-dimensional (3D) deconvolution using the measured point spread function (PSF) method. A representative plane is shown. (B) The relative amount of Lck at the plasma membrane of cells from panel A was quantified. Deconvolved z-stacks from 15 cells obtained from 3 separate experiments were used for fluorescent intensity measurements as described in “Methods.” The integrated fluorescent intensity (IFI) of Lck at the plasma membrane (PM) was expressed as a percentage (mean ± SD) of the IFI of the entire cellular Lck. *P < .05 (Mann-Whitney U test). (C) Normal CD4 T cells were infected and sorted as in Figure 3A. Cells were immunostained for Lck (red) and Rab11 (blue). Z-stacks of green (GFP), red, and blue fluorescence were collected. Z-stacks of red and blue fluorescence were subjected to 3D deconvolution as in panel A. The boxed area in the overlay image is magnified and displayed to the right of the image. Pink areas in cells denote colocalization of red Lck and blue Rab11. (D) Deconvolved Z-stacks of Lck fluorescence from panel C were quantified as in panel B. Images of 15 cells from 3 experiments (each experiment done using blood from a different healthy donor) were measured. P < .05 (Mann-Whitney U test) for difference between G22V and EV cells (*), G22V and WT cells (#), and WT and EV cells ($).