Fig. 5. Y to F substitutions in the intracellular domain of VEC at positions 645, 731 or 733 affect lymphocyte migration in a dominant manner. GPNT cells were nucleofected with wt or Y to F mutants of pEGFP-N'-VEC. On average 80% of cells expressed VEC-EGFP over a period of 3-4 days. (A) Transfected GPNT cells were fixed after 2 days and VEC-GFP distribution was analyzed by fluorescent microscopy. Bar, 50 µm. (B) Three days after transfection, GPNT cells were fixed and VEC-GFP was expression analyzed using confocal microscopy. Bar, 10 µm. (C) Nucleofected GPNT cells were grown to confluence for 24-48 hours at which point equal expression was verified by fluorescent microscopy (see A). Lymphocyte adhesion (white) and migration (black) were then measured as described in Fig. 4. (D) Mouse VEC-null endothelioma cells (see Fig. 4) were nucleofected with wt or the indicated Y to F mutants of pEGFP-N'-VEC before T cell adhesion and migration was assessed. Significant differences were determined by Student's t-test (^*P<0.005; ^**P<0.0001).