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. 2006 Nov 27;203(12):2703–2714. doi: 10.1084/jem.20051730

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Copyright © 2006, The Rockefeller University Press

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Figure 5. — JAM-C regulates Rap1 activity and VE-cadherin–mediated contacts. (A) Pull-down assays were performed to isolate active GTP-bound Rap1 or Ras. Western blots for Rap1 or Ras revealed increased active Rap1 in HDMECs transfected with siRNA against JAM-C compared with the HDMECs transfected with control siRNA, whereas active Ras was unchanged. In contrast, no change in the total Rap1 or Ras level was observed. The insert shows densitometric analysis of the ratio between active and total Ras or Rap1 in HDMECs transfected with siRNA against JAM-C (gray bars) or with control siRNA (open bars). Data are shown relative to control (active GTPase/total GTPase in HDMECs transfected with control siRNA was set as 1) and are mean ± SD of three separate experiments. (B) The adhesion of HDMECs transfected with control siRNA (open bars) or HDMECs transfected with siRNA against JAM-C (gray bars) to immobilized BSA, control Fc protein, Fc–VE-cadherin, or Fc–PECAM-1 is demonstrated. Adhesion is shown as the percentage of adherent cells. Data are mean ± SD of a typical experiment; similar results were obtained in three separate experiments. (C) The expression of VE-cadherin was analyzed by Western blot in HDMECs transfected with siRNA against JAM-C or with control siRNA as indicated.