Anti ve cadherin

For immunostaining: anti-CD31 (Biocare medical, CM303B; 1:200), anti-Ki67 (Thermo, RM9106S1; 1:200), anti-VE-Cadherin (BioLegend, 348,501; 1:100), anti-Erg1/2/3-Alexa Fluor 647 (Santa Cruz, sc-376293; 1:100), and anti-SMA-Cy3 (Sigma, C6198; 1:200) antibodies were used. For western blotting: anti-BMP10-GFD (R&D systems, MAB6038; 1:1000), anti-BMP10-Pro (R&D systems, AF3956; 1:1500), and anti-TNNI3 (abcam, ab56357; 1:3000) antibodies were used.

U87MG and HUVEC cells were seeded on Matrigel^® for Tube Assay as previously described; tubes were fixed with 4% PFA solution for 20’ at room temperature. The fixed tubes were treated with PBS 10% FBS blocking solution for 1 h at room temperature and incubated with monoclonal I antibodies anti VE-cadherin (BioLegend Inc., San Diego, CA, USA) (1:250) or anti-CD31 (BD Biosciences Pharmingen, San Diego, CA, USA) for 3 h at room temperature. The samples were then incubated with II Ab anti mouse IgG HRP conjugated (ImmunoReagents Inc., Raleigh, NC, USA) (1:5000 in PBS) for 1 h at room temperature. Antibody positivity was revealed by peroxidase substrate DAB substrate Kit (Vector Laboratories, Burlingame, CA, USA). Cells were observed by a light inverted microscope at 10× magnification. Negative controls were incubated with anti-mouse IgG only.