AKAP9 depletion does not affect Rap activation, VE-cadherin adhesion, or organization of cortical actin. Control and AKAP9 (Akp9) siRNA HUVECs were evaluated for Rap1 GTPase activity (A); microtubule distribution, actin organization, and VE-cadherin linearity at junctions (B); and VE-cadherin homophilic interactions (C), following 20 minutes of treatment with vehicle control (−) or O-Me-cAMP (+). (A) Cell lysates were subjected to pull-down assays to detect active Rap1 (Rap1GTP). O-Me-cAMP similarly enhanced Rap activation in both control and AKAP9 siRNA-treated monolayers. Western blot of total Rap1 (total Rap) shows equivalent levels of Rap1 GTPase in all samples. (B) Cells were fixed and stained for microtubule (α-tubulin), actin (rhodamine-phalloidin) or VE-cadherin. O-Me-cAMP treatment of control siRNA cells resulted in microtubule extension to the periphery of the cell (*) compared with vehicle-treated cells following O-Me-cAMP treatment, while the MTs in AKAP9-silenced cells were disorganized under either condition. In contrast, AKAP9 silencing had no effect on the ability of O-Me-cAMP to increase cortical actin bundles (arrow, middle panels) or to enhance the linearity of VE-cadherin staining (arrow, bottom panel). Scale bar = 10 μm. (C) Vybrant CFDA-labeled cells were plated on VE-cadherin Fc-coated dishes without (Control) or with O-Me-cAMP (O-Me) treatment, and cell adhesion was quantified by fluorometric analysis. (D) Analysis of de novo AJ formation. Cells were cultured in complete medium (Control), placed in low-Ca²⁺ medium (−Ca²⁺), and then in medium replenished with Ca²⁺ (+Ca²⁺). Cell samples were fixed at these different phases and immunostained for VE-cadherin, β-catenin, or p120 as indicated, and fluorescence intensity at junctions was determined. (E) Analysis of dynein distribution. Control and AKAP9 siRNA cells were subjected to the Ca²⁺ switch assay, as described in panel D, and stained for dynein. Dynein was initially absent at the junctions of cells, but similarly appeared at cell-cell contacts (arrow) of control and AKAP9 siRNA cells following Ca²⁺ replenishment (+Ca²⁺). n = 3 for experiments in panels A through E.