Noc 18

Anti-VEGFR2, anti-PLCγ, anti-HSP90, anti-Raf-1, anti-cyclin D1, anti-CDK4 and anti-p21 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-VEGFR2, anti-phospho-VEGFR2 (Y1175), anti-phospho-PLCγ (Y783), anti-phospho-Erk (T202/Y204), anti-Erk, antiphospho-Akt (S473) and anti-Akt antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-eNOS and anti-phospho-eNOS (S1177) antibodies were purchased from BD Biosciences (San Diego, CA, USA). Anti-α-tubulin antibody was purchased from Sigma-Aldrich. Recombinant human VEGF165 and 4,5-diaminofluorescein diacetate (DAF2-DA) and DAPI (4′,6-diamidino-2-phenylindole dihydrochloride hydrate) were purchased from Sigma-Aldrich. DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) and NOC-18 was purchased from Santa Cruz Biotechnology.

Anti-eNOS (ab5589) and anti-iNOS (ab3523) used for immunohistochemistry were from Abcam (Paris, France). Anti-eNOS antibody (AF950) used for immunoprecipitation experiments was from R&D Systems (Bio-Techne, France). Anti-glutathione antibody recognizing GS-S-proteins was from Virogen (Watertown, MA, USA). Secondary antibodies anti-mouse and anti-rabbit HRP-conjugated were from Cell Signaling Technology (Ozyme, France). Anti-Von Willebrand Factor (VWF) (AB7356) was from Chemicon (Merck Millipore) and anti-VEGF was from Sigma. Secondary anti-goat HRP-conjugated was purchased from Southern Biotech (Clinisciences, France). Secondary Alexa Fluor antibodies (488 and 546) were from Life Technologies (Courtaboeuf, France). Dihydroethidine (DHE), DAF-FM diacetate (4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate), dithiotreitol (DTT), 4,6-Diamidino 2-phenylindole dihydrochloride (DAPI), oxypurinol, VAS2870, L-NAME (N-Nitro-L-arginine methyl ester hydrochloride), BH4 (tetrahydrobiopterin dihydrochloride) were from Sigma-Aldrich (Saint Quentin Fallavier, France). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) and SYTO-13 were from Thermofisher (Villebon sur Yvette, France), NOC-18 (diethylenetriamine/nitric oxide adduct; DETA NONOate), was from Santa Cruz Biotechnology (Clinisciences, France).

Normal human astrocytes and neurons grown to confluence were stimulated with the nitric oxide donor, NOC-18 (100 μM; Santa Cruz, CA, United States), tumor necrosis factor alpha, TNF-α (100 nM; Life Technologies, Carlsbad, CA, United States), or the stable ATP analog, ATP-γ-S (100 μM, Sigma, MS) for 24 h. The cultures were fixed and washed/permeabilized with 0.1% Triton and processed for quantitative immunofluorescence.