Ve cadherin

Mouse ephrin-A1 (R&D Systems), VE-cadherin (Abcam), and E-cadherin (Abcam) ELISA kits were used to detect the presence of soluble ephrin-A1 ligand, VE-cadherin, and E-cadherin, respectively, in the plasma of mice. Mouse ephrin-A1 ELISA kits were also used to detect soluble ephrin-A1 present in MBMEC culture supernatants post-stimulation as described in detail in a previous section. Mice were euthanized with isoflurane and whole blood was collected into heparinized tubes and centrifuged at 1000g for 15 minutes at 4°C to isolate plasma. Plasma was diluted 1:10 for ELISA analysis of ephrin-A1 ligand, 1:800 for ELISA analysis of VE-cadherin, and 1:2000 for ELISA analysis of E-cadherin. Absorbance was measured at 450nm using a plate reader (Biotek) and plasma concentrations were determined using standard curves as per the manufacturer’s instructions. Human ephrin-A1 (Sino Biological) and human EphA2 (R&D Systems) ELISA kits were used to detect the presence of soluble ephrin-A1 ligand and soluble EphA2, respectively, in the plasma of humans and carried out according to the manufacturer’s instructions.

As important markers for endothelial barrier function, the distribution of the tight junction scaffolding protein ZO-1 and adherens junction protein VE-cadherin were observed (36 (link)). After relevant treatment, GEnCs were washed in PBS and fixed with 4% formaldehyde for 30 min. Next, the GEnCs were permeabilized with 0.5% Triton X-100, washed and blocked with 5% BSA for 1 h at room temperature. After incubation with primary antibodies (ZO-1, dilution 1/100, Life, Carlsbad, CA, USA; VE-cadherin, dilution 1/200, Abcam, Cambridge, MA, USA) at 4°C overnight and a thorough wash in PBS, the GEnCs were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (for the detection of ZO-1, dilution 1/200, Jackson ImmunoResearch, West Grove, PA, USA; for the detection of VE-cadherin, dilution 1/500, Abcam, Cambridge, MA, USA) at 37°C for 1 h. Eventually, the specimens were stained with 10 μg/ml 4',6-diamidino-2-phenylindole (DAPI) and mounted with Mowiol. The immunofluorescence staining was photographed by a fluorescence microscope (Nikon Eclipse 90i, Nikon Instruments Inc., Tokyo, Japan). At least 10 visual fields per slide of GEnCs at ×400 were observed blindly. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to evaluate the immunofluorescence staining of ZO-1 and VE-cadherin. Positive signals were quantified as signal intensity.

Protein levels of the lungs were measured by western blot. The frozen right lungs were mechanically disrupted and the total protein lysates were extracted using protein lysates (CST). Equal amounts of protein were separated by SDS/PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Following blocking with 5% bovine serum albumin for 1h at room temperature, the membranes were incubated at 4 °C overnight with a primary antibody recognizing VE-cadherin (HuaAn, Hangzhou, China), β-catenin (HuaAn), phosphorylated-β-catenin (Thr 41/Ser 45) (HuaAn), Src (JF0947) (HuaAn), phosphorylated—Src (Y529) (Abcam, Cambridge, UK), gsk3β (HuaAn), phosphorylated-gsk3β (Y216 + Y279) (Abcam), PI3K (HuaAn) and β-actin (HuaAn). Then the membranes were incubated with secondary antibodies coupled to horseradish peroxidase at room temperature for 1 h and detected using a Tanon-4500 gel imaging system.