Pyk2 activity regulates VE-cadherin/VE-PTP dissociation. (A) Pyk2 inhibition prevents VE-cadherin/VE-PTP dissociation. TNF-α–stimulated bEnd.5 cells were preincubated with the indicated concentrations of the Pyk2/FAK inhibitor PF431396, followed by the incubation with or without T cells and subsequent VE-PTP/VE-cadherin coimmunoprecipitation analysis. (B) FAK activity is not required for VE-cadherin/VE-PTP dissociation. Analysis done is analogous to panel A, comparing the inhibitory activity of the dual-specificity Pyk2/FAK inhibitor PF431396 (5 μM) and the monospecific FAK inhibitor PF573228 (5 μM). (C) Pyk2 acts downstream of NOX activation. TNF-α–stimulated bEnd.5 cells were preincubated with the NOX inhibitor VAS2870 (10 μM) and were subsequently stimulated with or without anti–VCAM-1 mAb-loaded beads (VCAM XL), followed by immunoprecipitation of Pyk2 and immunoblotting with a general antiphosphotyrosine mAb (WB: pTyr), antibodies against phosphorylated tyrosine 402 of Pyk2 (WB: pPyk2 [Tyr402]), or antibodies against Pyk2 (WB: Pyk2). Equivalent aliquots of total cell lysates were analyzed by immunoblotting for Pyk2 (WB: Pyk2 total lysate).