Nef mutants defective for association with Pak2 are also defective for the ability to enhance T-cell activation upon PHA stimulation. A) Jurkat E6-1 cell lysates were harvested at 48 hpi following transduction with VSV-G pseudotyped pHAGE-Nef-HA-IRES-ZsGreen vector virions encoding wild-type (Lane 2) or mutant Nefs (Lanes 3 and 4), or empty vector (Lane 1), separated by SDS-MOPS-PAGE, and immunoblotted to detect HA-tagged Nef and endogenous Pak2. Lane 4 is a non-contiguous lane from the same gel as Lanes 1–3. For clarity, brightness and contrast of the Nef panels were increased by 2%. B) CD25 expression on Jurkat E6-1 cells transduced with VSV-G pseudotyped pHAGE-EF1α-IRES-ZsGreen vector encoding wild-type (NL4-3 or 5C) or mutant (5C-7, 5C-AxxA) Nef or empty vector was analyzed at 48 h post-transduction, with (lower panel) or without (upper panel) 1 μg/mL PHA-P stimulation for 24 h prior to FACS analysis. ZsGreen expression is a reporter for pHAGE transduction and Nef expression, except in the case of vector samples, which lack Nef. For each sample, the Nef allele encoded in the pHAGE-IRES-ZsGreen vector virions is indicated (see legend). Bars graphed on the left and right of each panel represent the ZsGreen-negative and ZsGreen-positive subsets of cells from each transduction. C) FACS plots and gating strategy for panel B are shown. The gate “P2” demarcates populations of matched ZsGreen fluorescence for which percentages of CD25-positive cells are reported in panel B.