Phospho-VE-Cadherin (Tyr731) Antibody - #AF3265

Fig. 4 Overexpression of Robo4 improves IR-induced downregulation of VE-cadherin and decreases the permeability of the EC monolayer. A Confirmation of Robo4 overexpression after establishing a stable cell line using western blot and qPCR analysis, respectively. B Effect of Robo4 overexpression and irradiation on cell survival and proliferation of ECs. C and E Functional analysis of Robo4 overexpression by FITC-dextran (70 kDa) permeability assay and EC monolayer crystal violet staining after irradiation. D Western blot analysis of total and pY-731 VE-cadherin after Robo4 overexpression and irradiation; the relative protein expression of phosphorylated Y-731 and total VE-cadherin normalized with endogenous beta-actin protein; The ratio of pY-731 VE-cadherin to total VE-cadherin. Cycloheximide treatment and immunoblotting were conducted as in (F). The CD144 band intensity was normalized to actin and then normalized to the t = 0 controls. G Confocal microscopic images of VE-cadherin (red) and DAPI (blue). H Western blot analysis showing connexin 43 expression levels in Robo4 overexpression microvascular ECs after gamma radiation treatment. J Confocal images show immunofluorescence of connexin 43 (red) in Robo4 overexpression microvascular ECs, with or without irradiation. Nuclei appear in blue. I Flow cytometric analysis to determine EC GJ coupling ability in vitro. For all experiments, scale bars = 20 µm, n ≥ 3, and error bars represent std.

Fig. 4 (a) Schematic showing the protein pull down experimental setup. Immunoblotting (left panel) and its semi-quantitative analysis (right panel) of (b) VEC, SOD1, claudin-5 and α-tublin from whole cell lysate and pulled down by different NPs. (c) Y658 (p-VEC(Y658)), Y731 (p-VEC(Y731), VEC and α-tublin from the whole cell lysate with/without the Src kinase inhibitor, PP1.

Fig. 3. YAP inhibits VEGF expression to mediate endothelial barrier repair. (A–E) HUVECs were transfected with control scrambled (scr) or YAP shRNA and stable cell lines were screened. Cells were treated with TNF-α for 24 h. VEGF (A,B) and VE-cadherin (D,E) protein levels were analyzed by western blotting. Relative levels of VEGF (C) mRNA were determined by qRT-PCR. (F–I) HUVECs were transfected with GFP or YAP, and stable cell lines were screened. Cells were treated with TNF-α for 24 h. VEGF (F,G) and VE-cadherin (F,H) protein levels were assessed by western blot analysis. Relative levels of VEGF (I) mRNA were determined by qRT-PCR. All experiments were repeated three times (n = 3), and data are presented as means ± S.E. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2 Phosphorylated vascular endothelial (p-VE)-cadherin protein expression level is markedly increased in mouse aortic endothelial cells (MAECs) cultured in high glucose (HG) for 7 days and in thoracic aorta endothelial cells of streptozotocin (STZ)-induced mice with diabetes with no change in VE-cadherin expression level; HG exposure promotes intercellular permeability in MAECs. Representative western blot analysis images (A) and summary data (B) showing p-VE-cadherin and VE-cadherin expression levels in MAECs cultured in normal glucose (NG) or HG medium. (C, E) MAECs were cultured in HG medium for 7 days and then fixed and incubated with anti-p-VE-cadherin antibody (red), anti-VE-cadherin antibody (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue; cell nuclei) and then imaged with a confocal microscope. Representative confocal microscopy images and the final merged images are shown (C, E). (D, F) Fluorescence intensity profiles and summary data for anti-p-VE-cadherin antibody and anti-VE-cadherin antibody in the regions delineated by the corresponding yellow line shown in (C) and (E). (G) Representative images of thoracic aorta endothelium immunostaining (brown; eg, red arrowheads) for expression levels of p-VE-cadherin and VE-cadherin in a mouse model of diabetes mellitus (DM) and control mice. Magnification, ×200. (H) Quantification of p-VE-cadherin and VE-cadherin expression levels in the thoracic aorta endothelium of a mouse model of diabetes and control mice. IOD, integrated optical density. (I) HG-induced transendothelial electrical resistance (TER) was examined in vitro after MAECs were cultured in HG medium for 7 days. (J) FD-20 permeability was tested in a monolayer of aortic endothelial cells using a transwell permeability assay. OD, optical density; values, means±SEM (n=4–8 samples). *P<0.05, ***p<0.001 compared with NG-cultured cells or control groups.

Figure 2 Phosphorylated vascular endothelial (p-VE)-cadherin protein expression level is markedly increased in mouse aortic endothelial cells (MAECs) cultured in high glucose (HG) for 7 days and in thoracic aorta endothelial cells of streptozotocin (STZ)-induced mice with diabetes with no change in VE-cadherin expression level; HG exposure promotes intercellular permeability in MAECs. Representative western blot analysis images (A) and summary data (B) showing p-VE-cadherin and VE-cadherin expression levels in MAECs cultured in normal glucose (NG) or HG medium. (C, E) MAECs were cultured in HG medium for 7 days and then fixed and incubated with anti-p-VE-cadherin antibody (red), anti-VE-cadherin antibody (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue; cell nuclei) and then imaged with a confocal microscope. Representative confocal microscopy images and the final merged images are shown (C, E). (D, F) Fluorescence intensity profiles and summary data for anti-p-VE-cadherin antibody and anti-VE-cadherin antibody in the regions delineated by the corresponding yellow line shown in (C) and (E). (G) Representative images of thoracic aorta endothelium immunostaining (brown; eg, red arrowheads) for expression levels of p-VE-cadherin and VE-cadherin in a mouse model of diabetes mellitus (DM) and control mice. Magnification, ×200. (H) Quantification of p-VE-cadherin and VE-cadherin expression levels in the thoracic aorta endothelium of a mouse model of diabetes and control mice. IOD, integrated optical density. (I) HG-induced transendothelial electrical resistance (TER) was examined in vitro after MAECs were cultured in HG medium for 7 days. (J) FD-20 permeability was tested in a monolayer of aortic endothelial cells using a transwell permeability assay. OD, optical density; values, means±SEM (n=4–8 samples). *P<0.05, ***p<0.001 compared with NG-cultured cells or control groups.

Figure 2 Phosphorylated vascular endothelial (p-VE)-cadherin protein expression level is markedly increased in mouse aortic endothelial cells (MAECs) cultured in high glucose (HG) for 7 days and in thoracic aorta endothelial cells of streptozotocin (STZ)-induced mice with diabetes with no change in VE-cadherin expression level; HG exposure promotes intercellular permeability in MAECs. Representative western blot analysis images (A) and summary data (B) showing p-VE-cadherin and VE-cadherin expression levels in MAECs cultured in normal glucose (NG) or HG medium. (C, E) MAECs were cultured in HG medium for 7 days and then fixed and incubated with anti-p-VE-cadherin antibody (red), anti-VE-cadherin antibody (red) and 4',6-diamidino-2-phenylindole (DAPI) (blue; cell nuclei) and then imaged with a confocal microscope. Representative confocal microscopy images and the final merged images are shown (C, E). (D, F) Fluorescence intensity profiles and summary data for anti-p-VE-cadherin antibody and anti-VE-cadherin antibody in the regions delineated by the corresponding yellow line shown in (C) and (E). (G) Representative images of thoracic aorta endothelium immunostaining (brown; eg, red arrowheads) for expression levels of p-VE-cadherin and VE-cadherin in a mouse model of diabetes mellitus (DM) and control mice. Magnification, ×200. (H) Quantification of p-VE-cadherin and VE-cadherin expression levels in the thoracic aorta endothelium of a mouse model of diabetes and control mice. IOD, integrated optical density. (I) HG-induced transendothelial electrical resistance (TER) was examined in vitro after MAECs were cultured in HG medium for 7 days. (J) FD-20 permeability was tested in a monolayer of aortic endothelial cells using a transwell permeability assay. OD, optical density; values, means±SEM (n=4–8 samples). *P<0.05, ***p<0.001 compared with NG-cultured cells or control groups.

Figure 3. |BMP-2 increased the HUVECs permeability(A and B) VE-cad, p-VE-cad, and Occludin protein levels in HUVECs were determined (n=5).