Fig. 6.

Partial knockdown of TIAM1, in eNOS-deficient cells, restores the phosphorylation of VE-cadherin and the recruitment of c-Src to cellular junctions upon VEGF stimulation. (A) HDEMCs transfected with siRNA control, or siRNA against eNOS or eNOS and TIAM1 were serum starved and stimulated with VEGF (100 ng/ml) for 10 minutes. VE-cadherin was immunoprecipitated and the associated proteins were analyzed by immunoblotting against phosphorylated tyrosine (pY; 4G10, PY20 clone) and VE-cadherin. The whole cell lysates (WCL) were analyzed by western blotting for VE-cadherin, eNOS, TIAM1 and β-actin expression before the incubation with anti-VE-cadherin antibody. (B) HDEMCs transfected with eNOS siRNA or control siRNA, or with eNOS siRNA and TIAM1 siRNA, were co-labeled for VE-cadherin (green) and c-Src (red) after before and incubation with VEGF (100 ng/ml) for 15 minutes. The images have been captured with a Zeiss Axiovert epifluorescence microscope and a 63× oil immersion objective, using Openlab software (Improvision, Lexington, MA). Arrows depict c-Src recruitment to the plasma membrane, which colocalizes with VE-cadherin. Data are representative of three independent experiments.