Nef affects intracellular distribution of catalytically active Lck. (A) Western blot analysis of Jurkat T lymphocytes expressing Lck.GFP with RFP or Nef.RFP. All samples were generated within the same experiment and run on the identical gel but are shown as individual boxes to illustrate that lanes in between those shown were removed. Total and active Lck was detected by Abs that recognize all Lck species or are specific for the indicated Lck phospho-species, respectively. (B) LI-COR quantification of the Western blots shown in panel A. Depicted are the ratios of phosphorylated versus total Lck signal intensity. Note that the presence of Nef does not affect abundance of both phosphorylated Lck forms investigated. Values are the arithmetic means of at least 3 independent experiments ± SD (C) Pixel quantification of total or active Lck populations in the presence of the indicated GFP fusion proteins. Jurkat T lymphocytes expressing the indicated proteins were subjected to z-stacks of confocal microscopy, and the total amounts of Lck per cell were determined with ImageJ Version 1.42q software. Values for individual cells are presented with the arithmetic mean indicated by the black line, and are relative to ratios obtained for neighboring untransfected cells that were arbitrarily set to 1. (D) Representative confocal micrographs of Jurkat T lymphocytes expressing the indicated GFP proteins. Shown is the staining for the indicated phosphorylated Lck species. Asterisks indicate GFP-positive cells. Scale bar indicates 10 μm. (E) Numbers of cells that display RE/TGN accumulation of total and active Lck populations. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 cells were counted per condition. (F) Magnitude of Lck RE/TGN targeting per cell. Depicted are the percentages of Lck signal per cell in RE/TGN compartments. Values for individual cells are presented, with the arithmetic mean indicated by the black line.