PAK2 binding is not essential and downregulation of CD3 is sufficient to hamper T-cell development by SIV Nef. Flow cytometric analysis of FTOC initiated with retrovirally transduced CD34+ thymic progenitors. (A) Dot plots show eGFP vs. CD4 and eGFP vs. CD3 staining gated on live human cells of Nef transduced cultures, as indicated. Quadrants were set to include 99% of non-transduced cells stained with isotypic controls in lower left quadrant. Numbers in plots indicate mean fluorescent intensity for the marker on the Y-axis (CD4 or CD3) for all eGFP- cells (upper and lower left quadrants) or all eGFP+ cells (upper and lower right quadrants) are indicated. (B) T-cell generation ratio calculated from Nef transduced cultures, as indicated. Bars represent averages of at least 3 independent experiments, error bars indicate standard deviation. Nef proteins that bind PAK2 and downregulate CD3 are shown in dark grey, mutants showing reduced PAK2 binding in blue, mutants with no detectable PAK2 binding in red, mutants showing no detectable PAK2 binding nor CD3 downregulation in black. Difference compared to control eGFP (white bar) was tested with Mann–Whitney U test, *p < 0.05, **p < 0.005, ***p < 0.0005.