Fig 1. Marked accumulation of phosphotyrosine-containing proteins in the apical membrane domain of endothelial cells forming lumens and tubes.

(A) ECs were suspended in 3D collagen matrices and after the indicated times were fixed and immunostained with anti-phosphotyrosine antibodies. Cells were also stained for F-actin using AlexaFluor-conjugated phalloidin, Hoescht dye to label nuclei and were imaged using confocal microscopy. Bar equals 20 μm. (B) ECs were seeded in 3D collagen matrices in the presence or absence of the Src inhibitor, PP2 at 10 μM. After 24 hrs, cultures were fixed, stained with toluidine blue and quantitated for lumen formation. Data are reported as the mean total vessel area per high-power field (HPF) ± standard deviation (SD) (n = 15, p < 0.01). Asterisks indicate significance compared to control cultures. Bar equals 100 μm. EC cultures were established and were fixed at 12 hr (C) or 16 hr (D) and were stained in the same manner as described in (A). Arrowheads indicate apical membrane staining; arrows indicate the basal distribution of F-actin staining. L indicates lumen, while v indicates intracellular vacuoles. Bar equals 25 μm.