KIR2DL1 recruits SHP-1 and/or SHP-2 differently, depending on whether it is bound to its ligand. (A) Total protein lysates from JK32 and EV cells were blotted with anti-GFP mAb: KIR2DL1-GFP fusion protein and GFP are identified at 85 and 27 kDa, respectively. (B) EV and JK32 T cells were stimulated with SEE-pulsed 721.221/HLA-Cw4 or 721.221/HLA-Cw7 cells for 20 minutes and lysed, and GFP was immunoprecipitated. GFP immunoprecipitates (IP) were analyzed by immunoblotting with antiphosphotyrosine (p-Tyr) antibodies, or (C) the membrane was probed with anti–SHP-1 and anti–SHP-2 mAbs. (D) The distribution of SHP-1 and SHP-2 (red fluorescence) was analyzed by confocal microscopy, as detailed in Figure 4. KIR2DL1 appears as green fluorescence. (E) Cells were treated as described in panel D, and images of 5 fields were visualized with a 63×/1.4 oil-immersion objective. Signal intensity was quantified with ImageJ software.