VE-cadherin expression and phosphorylation in human glioma tissues.

<p>(A,B) Representative (n=10 samples) micrographs of primary human glioma stained for VE-cadherin (in red; nuclei are stained in blue). Images were processed using Adobe Photoshop Scale bar: 25 μm. A capillary network positive for VE-cadherin was detected in all the tumors. (C) A 125 kDa fragment of VE-cadherin was highly detectable in glioblastoma (GBM) extract and not in non-tumor brain tissue (N). (D) Protein lysates from N and GBM were analyzed by SDS-PAGE and western blotting with the antiphophotyrosine antibody (clone 4G10). Several proteins with apparent molecular masses (indicated by filled arrowheads) ranging from 220 to 25 kDa displayed clearly strong tyrosine phosphorylation in GBM but not in non-tumor tissue. (E) 500 μg of GBM tissue lysate protein were immunoprecipitated with an anti-human VE-Cadherin antibody directed to C-term of the protein and blotted with the indicated antibodies (Ptyr or VE-cad). Immunoprecipitation of VE-cadherin from glioma extracts allowed to detect a tyrosine phosphorylated form of VE-cadherin. Sample loading was controlled using actin detection. (F) Active Src (phosphoY418) was highly detectable in GBM but not in non-tumor (N) brain extract. (G) Orthovanadate treated-HUVECs lysates (control: CTL) and GBM extracts (50 µg) were analyzed by western blot using antiphosphosite antibodies directed against Y⁶⁵⁸ and Y⁷³¹, and the antibody directed against pY⁶⁸⁵ VE-cadherin raised in our laboratories. Only pY⁶⁸⁵ was detected in GBM as in HUVECs upon VEGF stimulation (50 ng/mL). (H) Same experiment as described in (E), and immunoblotting with anti-Csk antibody and anti-pY⁶⁸⁵ VE-cadherin antibody. The association of Csk with VE-cadherin in GBM confirmed the phosphorylation at the site Y⁶⁸⁵ also detected with the antiphosphosite antibody. Filled arrowhead indicates the position of the IgG heavy chains of the crosslinking antibody. In all blots the position of size markers (in kDa) is indicated on the left. These experiments were repeated at least three times in a similar configuration.