rTMD1 interacts with LeYAg. (A) SPR measurement. Various concentrations of LeY were passed through a HEK293-expressed rTMD1Δ-conjugated CM5 chip. The x-axis shows flow time, and the y-axis shows interaction strength. (B) Confocal microscopy image shows the distribution of LeY on HUVECs. (Left panel) The expression of LeY while HUVECs were grown on gelatin-coated surface; the inset shows the IgM control. (Right panel) The distribution of LeY on HUVECs during tube formation on Matrigel. Confocal microscopy was used to obtain an XY-Z stack (3-D series). (a) An X-Y frame shows the distribution of LeY on membrane protrusions. (b) A phase-contrast image (bright field) from same area as that in subpanel a. (c) Confocal microscopy image of transverse X-Z and Y-Z sections of HUVECs labeled with LeY Ab on Matrigel, respectively. (d) Confocal microscopy image of a 3-D reconstruction of HUVECs labeled with LeY Ab on Matrigel. Pseudo-color represents the strength of the signal. The strongest signal is displayed in white. (C) Confocal microscopy image shows the interaction of HEK293-expressed rTMD1-FITC and LeY on HUVECs during tube formation on Matrigel. HUVECs were dissociated in buffer from the culture dish by nonenzymatic cell dissociation and incubated with rTMD1-FITC (C) or BSA-FITC (D) on Matrigel for 1 hour. Green, rTMD1-FITC (C) or BSA-FITC (D); red, LeY; blue, nucleus (DAPI stain). Merged channel shows colocalization (white arrows) of rTMD1-FITC and LeY at membrane ruffles and protrusions. A similar result was obtained by assessing 25 cells by confocal microscopy. Representative figures are shown. Observations were repeated in at least 3 separate experiments.