ICAM-1 Clustering on Endothelial Cells Recruits VCAM-1
Figure 3
VCAM-1 associates with clustered ICAM-1. (a) Beads were coated with the indicated Abs as described in Section 2 and subsequently incubated with lysates from TNFα-treated HUVECs, followed by centrifugation, washing and analysis of bound proteins by SDS-PAGE and Western blotting. Blots were stained for VCAM-1 and show that VCAM-1 was precipitated with VCAM-1 beads and also with ICAM-1 beads. No VCAM-1 was detected in pull-downs using anti-IgG antibody coated beads. Lower panel shows expression of VCAM-1 in cell lysates as a loading control. Data are representative for three experiments. (b) The pull-down experiment was carried out as described under (a) and blots were stained for ICAM-1. ICAM-1 was precipitated with ICAM-1 beads, and a fraction of ICAM-1 associates with VCAM-1 beads, albeit less efficient than the reciprocal experiment shown in (a). No ICAM-1 was detected in pull-down experiments using anti-IgG coated beads. Lower panel shows expression of ICAM-1 as loading control in cell lysates. Data are representative for three experiments. (c) TNFα-stimulated HUVECs were incubated with ICAM-1 Ab in solution for 30 minutes and subsequently crosslinked with secondary antimouse Ab for an additional 30 minutes. Cells were lysed and ICAM-1 was immunoprecipitated and analyzed by Western blotting, which shows that ICAM-1-clustering induced association with VCAM-1. Lower panel shows ICAM-1 expression in cell lysates. Bottom panel shows that crosslinking of MHC class I protein did not induce any binding of VCAM-1. (d) ICAM-1 is precipitated by ICAM-1 beads, but not by anti-MHC class I antibody coated beads. Lower panel shows expression of ICAM-1 in cell lysates. (e) No ICAM-1 is precipitated by anti-PECAM-1 antibody coated beads (upper panel). Lower panel shows precipitation of PECAM-1 with the beads.
