Anti vegf

Brain samples were harvested and kept in 4% paraformaldehyde for 24 h and immersed in 30% sucrose for 3-4 days at 4°C. After cryoprotection in 30% sucrose, the brains were rapidly frozen in isopentane and stored at −80°C. Twenty micrometer cryostat sections at the level of the thalamus (bregma −3.3 mm) according to a stereotaxic atlas [15 ] were processed for immunohistochemistry. Immunohistochemistry analyses were performed with the UltraVision Quanto Detection System HRP DAB kit (Thermo Fisher Scientific, USA). The primary antibodies utilized were rabbit anti-GFAP antibody (glial fibrillary acidic protein, Santa Cruz Biotechnology, USA, 1 : 200 dilution), anti-VEGF (vascular endothelial growth factor, Millipore Corporation, USA, 1 : 400), and anti-MBP (myelin basic protein, Santa Cruz Biotechnology, USA, 1 : 200). The secondary antibodies utilized were biotinylated goat anti-mouse IgG and goat anti-rabbit IgG (Thermo Fisher Scientific, USA, 1 : 400 dilution). The results were observed using the Leica vertical microscope.

U-251 cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin and 0.1 μg/μl streptomycin, and incubated in 5% CO2 at 37°C. Cells were transfected in 6-well plates by Lipofectin (Invitrogen, Carlsbad, CA) using 0-100 nM GT198 siRNA as indicated. After 16 hours, the cytosolic fraction was isolated using lysis buffer (20 mM HEPES, pH 7.4, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 μg/ml protease inhibitors) with the addition of 1% Triton X-100 for 15 min on ice, and was probed by rabbit anti-VEGF (RB-9031) and anti-vWF. For the nuclear extract fraction, cells were lysed in the above lysis buffer with 0.05% Triton X-100 for 15 min on ice. The nuclei were then collected by centrifugation and further lysed in the above buffer with 420 mM NaCl for 30 min. Nuclear extracts were probed by anti-VEGF, anti-GT198, and anti-β-actin (Sigma, St Louis, MO). Western blots were detected using the ECL system (GE Healthcare, Piscataway, NJ). For reverse-transcription PCR, total RNA from siRNA-transfected U-251 cells was isolated by Trizol and analyzed using one-step RT-PCR kit (Qiagen, Valencia, CA). GT198 primers used are at Exon 1 and 5: 5’-CTTCCCCTTCAGCCAATCAC-3’ and 5’-GGTAGCTGCTTTAATGTTCTTCAA-3’.

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).

Cisplatin (DDP) was from Haosen Pharmaceutical Group Co., Ltd., Jiangsu, China. The total RNA extraction kit, reverse transcription kit, high-purity gel extraction kit, Western blotting test kit, and ECL test kit were all purchased from TaKaRa, Dalian, Liaoning, China. AnnexinV-FITC apoptosis kit was purchased from BD Company, BD Bioscience, San Jose, CA, USA. The secondary antibodies and the sheep anti-rabbit IgG were purchased from Sangon Biotech, Shanghai, China. Anti-VEGF, anti-survivin, anti-BCL-2, anti-BCL-xl, anti-cleaved caspase-3, and anti-cleaved caspase-9 antibodies were from Sigma, Newark, DE, USA. The rest of the reagents were from Invitrogen, MBI, Waltham, MA, USA.

Total protein was extracted with RIPA lysis buffer, and its concentration was measured using a BCA Protein Assay Kit (KGP902; KeyGen Biotech, Nanjing, China). Proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a nitrocellulose filter membrane. The membranes were incubated with blocking buffer consisting of 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBST), for 60 min at room temperature. After washing the membranes with TBST, they were incubated at 4°C overnight with the following primary antibodies: anti-MMP-2, anti-MMP-9, anti-E-cad, anti-VEGF, anti-Vimentin, anti-N-cad, or anti-GAPDH polyclonal antibodies (diluted 1 : 2000 in blocking solution; Sigma-Aldrich). An enhanced chemiluminescence reagent (KeyGen, Nanjing, China) was added after incubation with HRP-conjugated goat anti-rabbit IgG secondary antibody (1 : 5000 in blocking solution; Sigma-Aldrich) for 1 h at room temperature. A Bio-Rad gel imaging system was used for protein band visualization, GAPDH was used as an internal reference, and the relative protein expression level was calculated by the ratio of the gray value of the target protein to that of GAPDH.

The following primary antibodies were used for immunoblotting: anti-Osx antibody (Abcam); anti-Flag antibody (Sigma Chemical Co., St Louis, MO, USA); anti-MMP13, anti-VEGF, anti-CD34, and anti-PTHrP antibodies (Protech, Nanking Dist, Taipei, Taiwan); and anti-β-actin, anti-rabbit, anti-rat, and anti-mouse IgG antibodies (Bioworld Technology, Minneapolis, MN, USA).