Phospho-VE-Cadherin (Tyr658) Antibody - #AF8206

Figure 4. SARS‐CoV‐2 spike protein disrupts the vascular barrier via inhibiting endothelial VE‐cadherin A. The normalized fluorescence intensity of TRITC‐Dextran dye from co‐culture systems in which Caco‐2 cells were incubated with Control‐Fc or Spike RBD‐Fc after 24 h. For each group, n = 3. Data are from three independent biological experiments. B. The expression of junctional proteins ZO‐1, VE‐cadherin (VE‐cad), phosphorylated VE‐cad (pVE‐cad; 658), and pVE‐cad (731) in HUVECs co‐cultured with the supernatant of Caco‐2 cells that were treated with either Control‐Fc or Spike RBD‐Fc detected by Western blot. Protein expression was normalized to β‐actin. Data are from three independent biological experiments. C, D. Immunohistochemical staining shows levels of VE‐cad (C) and pVE‐cad (731) (D) in the duodenum tissue from COVID‐19 patients and healthy controls. Scale bars, 100 μm. For each group, n = 7. n, biologically independent samples (human specimen). E. Immunofluorescence analysis of VE‐cad expression and subcellular localization in Caco‐2 cells that were treated with Control‐Fc, Spike RBD‐Fc, or Spike RBD‐Fc combined with SCH772984 or Bevacizumab by confocal microscopy. VE‐cad staining in green and nuclear staining in blue. Scale bar, 20 µm. F. Fluorescence intensity of TRITC‐Dextran dye from co‐culture systems in which Caco‐2 cells were incubated with Control‐Fc, Spike RBD‐Fc, or Spike RBD‐Fc combined with SCH772984 or Bevacizumab. Data are from five technical replicates with similar results from three biological replicates. G. Immunohistochemical staining shows levels of VE‐cad and pVE‐cad (731) in the intestinal tissues of mice treated with Control‐Fc, Spike RBD‐Fc, or Spike RBD‐Fc combined with SCH772984 or Bevacizumab. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice).

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.

Figure 2. Exogenous administration of NMN protects endothelial cell barrier properties. HUVECs were placed in either normoxic conditions or a low oxygen-containing gas chamber (94% N2, 5% CO2, and 1% O2) for 24 hours of hypoxia treatment. NMN treatment meant dissolving NMN in the cell culture medium and incubating it 24 hours before hypoxia intervention, with NMN treatment in the normoxic group conducted simultaneously with the hypoxic group. (A) Showed the permeability of HUVECs monolayers (n = 3, independent experimental replicates), calculated as permeability = OD value of the lower chamber/(OD value of the upper chamber + OD value of the lower chamber). (B, C) Flow cytometry was used to detect apoptosis rates (n = 3, independent experimental replicates). Panel B shows the quantification results of panel C, where total apoptosis rate = Q2 value + Q3 value. In panel C, the X-axis represents the FITC channel, with positivity indicating Annexin-V staining of phosphatidylserine, and the Y-axis represents the PE channel, with positivity indicating propidium iodide (PI) staining of DNA. (D, E) Western blot analysis and quantification of phosphorylated and total VE-cadherin in HUVECs under different interventions (n = 3, independent experimental replicates). The signal for phosphorylated VE-cadherin was normalized to total VE-cadherin. The relative signal of phosphorylated VE-cadherin was normalized to the control group level (with each control group set to 1). β-Actin was used as a loading control. (F) Immunofluorescence staining was used to detect the expression of VE-cadherin (red) between HUVECs. Blue indicates DAPI staining. Scale bar = 20 µm. “Detail” shows a magnified view of the area within the yellow box in the image above. Values were expressed as the mean ± SD. One-way analysis of variance followed by Tukey's post hoc multiple comparison tests was used for panels A, B, and E. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.