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. 2004 May 17;199(10):1331–1341. doi: 10.1084/jem.20032206

Figure 5.

Figure 5.

PVR is the major DNAM-1 ligand on endothelial cells. (a) Inhibition of DNAM-1–Fc binding by different anti-Nectin mAbs. HUVECs, either untreated or preincubated with anti-PVR (L95) and anti–Nectin-2 (L14) mAbs used alone or in combination, were analyzed by two-color immunofluorescence and FACS® analysis for DNAM-1–Fc binding used at 1.5 μM. FITC-conjugated goat anti–mouse or PE-conjugated goat anti–human were used as second reagents. (b) Similar experiments were performed by immunofluorescence microscopy on confluent HUVECs. 1.5 μM DNAM-1–Fc binding at endothelial junctions was not affected by preincubation with anti–Nectin-1 (R1.302) or anti–Nectin-2 (L14) mAbs. Anti-PVR (L95) mAb preincubation strongly affects the DNAM-1–Fc binding at endothelial junctions. mAbs were used at 20 μg/ml. (c) HUVECs, preincubated with the different anti–Nectin-2 (gray bars) or anti-PVR (black bars) mAbs, were analyzed by FACS® analysis for DNAM-1–Fc binding used at 0.15 μM followed by FITC-conjugated goat anti–human second reagents. Untreated HUVECs stained with N4vtr-Fc were used as negative control (white bars). The value 100% corresponds to DNAM-1–Fc binding in the presence of the irrelevant anti–Nectin-1 (R1.302) mAb. (d) The different anti-PVR mAbs were mapped by ELISA using PVR-Fc molecules. PVR-vcc-Fc is constituted by the full-length ectodomain of PVR, whereas PVR-v-Fc is only constituted by the V domain of PVR.