VCAM-1 ligation dissociates VE-PTP from VE-cadherin. (A) VCAM-1 crosslinking (XL) induces VE-cadherin/VE-PTP dissociation. Either antigen-stimulated T cells or anti-VCAM-1–coupled dynabeads (mAb 6C7) were added to confluent TNF-α–stimulated bEnd.5 endothelioma cells for 5 or 15 minutes. VE-PTP was immunoprecipitated from lysates, and either coprecipitated VE-cadherin (WB: VE-cadherin) or precipitated VE-PTP (WB: VE-PTP) was detected by immunoblotting. To exclude proteolysis as a potential reason for the weakening of the VE-cadherin signal, aliquots of cell lysates were set aside and kept under identical conditions as lysates subjected to immunoprecipitations before direct analysis by immunoblotting (WB: VE-cadherin total lysate). (B) VCAM-1 crosslinking dissociates VE-cadherin and VE-PTP in murine primary endothelial cells. Confluent TNF-α–stimulated cells were incubated with anti-VCAM-1– or control IgG–coupled dynabeads for 15 minutes. Coprecipitated VE-cadherin was detected by immunoblotting as described above. (C) Anti–VCAM-1 blockade inhibits T-cell–induced dissociation. TNF-α–stimulated bEnd.5 cells were either incubated with control IgG– or anti-VCAM-1 antibody–loaded beads (first 2 lanes), or bEnd.5 cells were preincubated with anti-VCAM-1 antibody or no antibody. This was followed by incubation with T cells (last 2 lanes) and subsequent coimmunoprecipitation analysis. (D) ICAM-1 is not required for VE-cadherin/VE-PTP dissociation. TNF-α–stimulated bEnd.5 cells were either incubated with beads loaded with control IgG, anti–VCAM-1 mAb, or anti-ICAM-1 mAb (first 3 lanes), or the endothelial cells were preincubated with anti-VCAM-1 mAb or with anti-ICAM-1 mAb, followed by incubation with T cells (lanes 4-5) and subsequent coimmunoprecipitation analysis.