Anti ve cadherin

Protein was extracted from ASCs using RIPA lysis buffer (Beyotime) and then was quantified using a BCA Protein Assay Kit (Thermo Fisher). Ten to fifty microgram of each protein sample was separated via SDS-PAGE and electro-transferred onto PVDF membranes. Following blockade with PBST containing 5% BSA, the membranes were incubated with the following primary antibodies overnight at 4°C: anti-β-Actin (Santa Cruz); anti-TBX20 (sigma); anti-α-SMA (abcam); anti-Calponin (abcam); anti-SM22α (abcam); anti-Smad2 (HuaBio); anti-Smad3 (HuaBio); anti-phospho-Smad2 (HuaBio); anti-phospho-Smad3 (HuaBio); anti-CD31 (HuaBio); anti-VE-cadherin (R&D); and anti-VEGFR1 (HuaBio). The membranes were then washed four times with Tris-buffered saline/0.5% Tween-20 and incubated at 37°C for 1 h with the secondary antibodies (1:3,000; Thermo Fisher). Antigen and antibody complexes were detected by using an ECL Kit (Millipore). Immunoblots were quantified using Image Lab (version 6.0) software.

Differentiated ES cells were fixed with phosphate-buffered saline 4% paraformaldehyde (PBS-PFA) (Santa Cruz), treated for 1 h with PBS 0.1% Triton X100 (Merck Life Science) containing 5% donkey serum (Dako North America Inc) and 5% BSA (Merck Life Science) and incubated with primary antibodies for 3 h at 37 °C in a moist chamber. The following primary antibodies were used: anti-βIII-tubulin 1:1000 diluted in PBS (Merck Life Science) and anti-VE-cadherin 1: 100 diluted in PBS (R&D Systems). After rinsing three times with PBS, cells were incubated with 555 AlexaFluor donkey anti-goat and 488 AlexaFluor donkey anti-mouse (Invitrogen, Thermofisher Scientific) at 5 µg/ml for 1 h. After rinsing three times with PBS, nuclei were stained with DAPI and slides were sealed with aqueous mounting solution (Dako North America Inc). Images were captured by using a Leica TCS SPE confocal laser-scanning microscope, analyzed with Leica Confocal Software (LCS; Leica Microsystems). To quantify VE-cadherin⁺ endothelial cells, sequential images, covering the entire surface of a chamber slide well (0.7 cm²), were acquired with a Leica AF6000LX workstation. The area occupied by endothelial cells was measured with ImageJ software by using the Angiogenesis Analyzer for ImageJ [25 ].

HUVEC and ISO-HAS-B cells were cultured on gelatin coated coverslips and were either left untreated or transfected for ectopic expression of FoxO1 mutants. Cells were fixed for 10 min with 4% paraformaldehyde, washed with PBS and then permeabilized with 0.5% Triton-100 × -PBS for 10 min. Subsequently cells were blocked with 5% FCS 0.01% Tween-20-PBS for 30 min followed by incubation with anti-aPKC (1:100; Beckton Dickison), anti-VE-cadherin (1:100; R&D Systems); anti-c-Myc (1:100; Millipore); and anti-FLAG-M2 (1:400; Sigma) overnight at 4°C. Coverslips were then washed and incubated with the appropriate secondary antibodies (Alexafluor fluorescently conjugated; 1:400; Invitrogen) and Hoescht 33342 or DAPI, and mounted with Vectashield.