A novel signaling complex that couples spatial Rac1 activation to the NADPH oxidase. (A) VE-RC or VE-KO ECs were transiently transfected with HA-tagged Tiam1 expression construct. 24 h after transfection, cells were sheared for indicated times or kept as static control. Cell lysates were immunoprecipitated with an anti-HA antibody followed by immunoblotting with anti-Par3, anti–VE-cadherin, or anti-HA antibodies. Three experiments were performed for each cell type and numbers below the gel panels represent the fold of band intensity relative to t = 0 min. (B) VE-RC or VE-KO ECs were transiently transfected with HA-tagged Tiam1 and Flag-tagged p67phox. 24 h after transfection, cells were sheared for indicated times or kept as static control and cell lysates immunoprecipitated with anti-Flag M2 agarose. The immune complexes were subjected to SDS-PAGE followed by immunoblotting with anti-HA and anti-Flag antibodies. Three experiments were performed for each cell type and numbers below the gel panels represent the fold of band intensity relative to t = 0. (C) Tiam1 variants used in the in vitro direct binding assays. (D) Equal amounts of GST or GST-p67 fusions were incubated with indicated His-tagged Tiam1 variants in vitro. Bound proteins were detected by immunoblotting with an anti-6xHis monoclonal antibody. The white line indicates where the Western blot was cut to remove irrelevant lanes. (E) Shear stress regulates Rac1 activation through two distinct GEFs, Vav2 and Tiam1. Vav2 controls the global Rac1 GTP loading while Tiam1 links Rac1 to the flow-dependent polarity complex composed of Par3 and VE-cadherin. Tiam1 also couples the polarity complex to the NADPH oxidase for ROS production in a flow-dependent manner.