Figure 8.

Cytoskeletal organization and VE-cadherin endocytosis are not affected by VEC–α-C. (A) Immunofluorescence staining of VEC–WT and VEC–α-C endothelioma cells for vimentin, VE-cadherin and actin (as indicated). (B) Western blots of cell lysates of the same cells for total myosin light chain (MLC2) and its phosphorylated form. (C) F-actin and G-actin were isolated from the same cells and detected by immunoblotting. (D) Lung lysates of VEC–WT and VEC–α-C mice were immunoprecipitated for p120 or with control antibodies (Ctrl) and precipitates were immunoblotted for p120 or VE-cadherin. Bottom: respective immunoblots of total lung lysate aliquots. (E, F) Endocytosis of VE-cadherin. (E) VE-cadherin–GFP (VEC–WT) or VE-cadherin–α-catenin–GFP (VEC–α-C) was expressed by adenoviral vectors in HUVEC cells, incubated with an anti-VE-cadherin antibody at 4°C, and after washing away excess of antibody, cells were allowed to endocytose VE-cadherin for 0 or 30 min. Cells were fixed, permeabilized and stained with a secondary antibody against the first antibody (red). GFP is seen in green, merge to the right. (F) Quantification of intracellular (red) VE-cadherin staining of HUVEC that had endocytosed VE-cadherin–GFP (VEC–WT) or VE-cadherin–α-catenin–GFP (VEC–α-C) for 0, 15, 30 or 60 min, t-test. Figure source data can be found with the Supplementary Information.