Nef specifically prevents IS recruitment of active Lck but not of Fyn. (A) Representative micrographs of active Lck (p-Lck505&394) in the presence of the indicated GFP fusion proteins in T lymphocytes in conjugates with SEE-pulsed Raji B cells (in blue). Scale bar indicates 10 μm. (B) Frequencies of cells with pronounced RE/TGN accumulation (gray bars) or IS recruitment (black bars) of p-Lck505&394 on expression of the indicated GFP fusion proteins. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 conjugates were analyzed per condition. (C) Magnitude of RE/TGN accumulation (gray symbols) or IS recruitment (black symbols) of p-Lck505&394 per cell. Values for individual cells are presented, with the arithmetic mean indicated by the black line, and depict the percentage of total Lck signal per cell detected in RE/TGN compartments or at the IS, respectively (see also supplemental Figure 1B). (D) Representative micrographs of p-Fyn528 in the presence of the indicated GFP fusion proteins in T lymphocytes in conjugates with SEE-pulsed Raji B cells (in blue). Scale bar indicates 10 μm. (E) Frequencies of cells with pronounced RE/TGN targeting (gray bars) or IS recruitment (black bars) of p-Fyn528 on expression of the indicated GFP fusion proteins. Values are the arithmetic means of at least 3 independent experiments ± SD in which more than 100 conjugates were analyzed per condition. (F) Magnitude of RE/TGN targeting (gray symbols) or IS recruitment (black symbols) of p-Fyn528 per cell. Values for individual cells are presented, with the arithmetic mean indicated by the black line, and depict the percentage of total p-Fyn528 signal per cell detected in RE/TGN compartments or at the IS, respectively.