Rac1 activity is required for dissociation of VE-cadherin and VE-PTP. (A) Rac1 inhibition prevents VE-cadherin/VE-PTP dissociation. TNF-α–stimulated bEnd.5 cells were treated with the Rac1 inhibitor NSC23766 (25 μM) before incubation with antigen-stimulated T cells, followed by coimmunoprecipitation of VE-cadherin via VE-PTP (analogous to Figure 1). (B) Rac1 activation dissociates VE-PTP from VE-cadherin. bEnd.5 cells were incubated with a constitutively active mutant Rac1 fusion protein (Tat-Rac1-CA) or a wild-type fusion protein (Tat-Rac1-wt), followed by VE-PTP/VE-cadherin coimmunoprecipitation analysis. (C) VCAM-1 crosslinking (XL) activates Rac1. Anti-VCAM-1– or control IgG–coupled dynabeads or a Tat-Rac1-CA fusion protein (as a positive control) were incubated with TNF-α–stimulated bEnd.5 cells, followed by pulldown of active (GTP-bound) Rac1 with the purified Rac1-binding domain of PAK1 (as GST fusion protein). (D) TNF-α–stimulated bEnd.5 cells were incubated with or without T cells, and subsequently active Rac1 was analyzed as in panel C. For controls, GDP or nonhydrolysable GTPγS was added (right 2 lanes).