Ve cadherin

FACS analysis was performed using BD FACSAria II (BD Biosciences, San Diego, CA, USA) as described previously [9 (link), 17 (link)]. Stage specific embryos were separated from yolk sacs, and digested with 0.25% trypsin (Hyclone) to obtain a single cell suspension as previously described [11 (link)]. Cells were incubated with antibody cocktails for 30 minutes at 4°C, washed, and re-suspended in PBS with 2% FBS. Cocktails of antibodies CD31-APC (eBiosciences 25–0311), and VE-cadherin (BD Pharmigen) were used in this study, washed and re-suspended in FACS buffer (PBS/1%FBS). Cells were analyzed or sorted using FACSAria II (BD Biosciences). FACS data were quantified using data obtained from three independent experiments. AnnexinV-FITC labeling was performed according to the manufacturer’s protocol.

Cell lysates were centrifuged at 12,000 rpm for 7 min at 4 °C. Protein concentrations were determined using a Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, USA), and 10–15 μg of protein was loaded per lane onto 10% SDS–polyacrylamide gels, and the separated proteins were transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% bovine serum albumin (BSA) with 0.1% Tween-20 and incubated with the appropriate primary antibodies overnight at 4 °C ((fibronectin: BD Pharmigen), (CD31, VE-cadherin, β-actin, total and phospho-cofilin, total and phospho-cell division cycle (cdc) 42, Rho A, total and phospho-smad2/3: Cell Signaling, Danvers, MA, USA), (total and phospho-NF-κB p65, and total and phospho-inhibitor of NF (I)κBα: Santa Cruz Biotechnology, Dallas, TX, USA); (α-smooth muscle actin (SMA) and TGF-βR1: Abcam, Cambridge, UK)). The bands were later incubated with anti-rabbit or anti-mouse peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific). The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (Advansta, Menlo Park, CA, USA) and quantified using the ImageJ(1.52v, NIH)software.

The fluorescence microscopy analyses of VE-cadherin, β-catenin, and phospho-β-catenin Ser³³/Ser³⁷/Thr⁴¹were performed on the HUVECs in different conditions and times as indicated in Results. Briefly, after cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, cells were incubated with polyclonal antibodies against VE-cadherin (1:100; BD Transduction Laboratories), β-catenin (1:100; BD Transduction Laboratories), and phospho-β-catenin Ser³³/Ser³⁷/Thr⁴¹ (1:50; Cell Signaling Technology) overnight at 4°C, then washed and incubated with secondary Alexa Fluor–conjugated antibodies (1:300; Vector Laboratories) for 45 min at room temperature. After washing, the samples were mounted in VECTASHIELD (Vector Laboratories) and analyzed by fluorescence microscopy.