Anti vegfr2

2‐Phenylbenzimidazole‐5‐sulphonic acid (PBSA) was obtained from Sigma‐Aldrich. The structure of PBSA is presented in Figure 1A. The following chemical agents and antibodies were purchased from commercial sources: p38^MAPK inhibitor, SB203580 (Cayman Chemical); anti‐phospho‐Src (Y416), anti‐Src, anti‐phospho‐ERK (T202/Y204), anti‐phospho‐Akt (S473), anti‐phospho‐p70^S6K (T421/S424), anti‐phospho‐MKK3 (S189)/MKK‐6 (S207), anti‐MKK3, anti‐MKK6, anti‐phospho‐p38^MAPK (T180/Y182), anti‐phospho‐pRb (S780), anti‐phospho‐pRb (S807/S811), anti‐MMP‐2 and anti‐MMP‐9 (Cell Signaling); anti‐phospho‐FAK (Y397) and anti‐FAK (BD Biosciences); anti‐ERK, anti‐Akt, anti‐p70^S6K, anti‐38^MAPK, anti‐TIMP‐2, anti‐Cdk4, ant‐Cdk2, anti‐cyclin D, anti‐cyclin E, anti‐integrin β1, anti‐EGFR, anti‐FGFR‐1, anti‐VEGFR‐2, anti‐actin and mouse and rabbit IgG‐horseradish peroxidase conjugates (Santa Cruz Biotechnology Inc).

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-VEGFR2, anti-PLCγ, and anti-EEA1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-VEGFR2, anti-phospho-VEGFR2 (Y1175), anti-PECAM, anti-phospho-PLCγ (Y783), anti-phospho-Erk (T202/Y204), anti-Erk, anti-phospho-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, anti-ephrin-B2, and anti-Flag antibodies were purchased from Sigma-Aldrich. Anti-syntenin antibody was purchased from Abnova (Taipei City, Taiwan). Anti-mouse secondary Alexa 488 and anti-rabbit secondary Alexa 546 antibodies were purchased from Molecular Probes (Invitrogen). Recombinant human VEGF165 and 4,5-diaminofluorescein diacetate (DAF2-DA) were purchased from Sigma-Aldrich.

HUVECs were grown to confluence in 100-mm dishes and serum-starved for 12 h. Cells were pre-treated 1 h with vehicle (DMSO), curcumin (10 μM), or 1 (10 μM) at 37 °C and then stimulated with VEGF (10 ng/mL) for 10 min. Equal amounts of protein in cell extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 3% bovine serum albumin (w/v) in TBST (0.05 M Tris, 0.15 M NaCl, pH 7.5, and 0.1% Tween 20) at room temperature for 1 h, the membranes were immunoblotted using anti-phospho-ERK1/2, anti-ERK1/2, phospho-VEGFR2 (Cell Signaling Technology, Beverly, MA, USA), anti-VEGFR2, and anti-β-actin (Santa Cruz Biotechnology) antibodies, and then incubated with a horseradish peroxidase-conjugated secondary antibody. Immunoreactivity was detected using the Western Blotting Plus Chemiluminescence reagent (Thermo Scientific, Rockford, IL, USA). For tumor tissues, proteins were extracted by homogenizing the tissues. SDS-PAGE analysis and immunoblotting were performed as described above using anti-CD31 and anti-VEGFR2 antibodies.

Cells were harvested and lysed with RIPA buffer (Thermo Scientific Inc., Boston, MA, USA) containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and a phosphatase inhibitor cocktail (Sigma-Aldrich). Total protein concentrations were quantified using an RC/DC protein assay reagent (Bio-Rad, Hercules, CA, USA). Protein extracts were separated using 6 and 10% SDS-PAGE and transferred onto PVDF membranes (PALL, Westborough, MA, USA). Membranes were incubated in a blocking solution (Santa Cruz Biotech. Inc., Santa Cruz, CA, USA) for 30 min and incubated with anti-VEGF-A, anti-VEGFR-1, anti-VEGFR-2, anti-phospho FAK, anti-phospho ERK1/2, anti-phospho PI3K, anti-phospho AKT, anti-phospho JNK, anti-phospho p38 antibodies (Santa Cruz Biotech. Inc.) at 1:2000 dilution in a blocking solution overnight at 4 °C, and probed with peroxidase conjugated secondary antibodies at 1:5000 dilution. Protein bands were detected using enhanced chemiluminescent Western blotting detection reagent (Thermo Scientific Inc.)
Also, protein extracts were immunoprecipitated using a mouse anti-phospho-Tyr antibody (Santa Cruz Biotech. Inc.) and an ImmunoCruz™ IP/WB Optima kit (Santa Cruz Biotech. Inc.). Immunoprecipitated proteins were subjected to 6% SDS-PAGE and Western blotting using anti-VEGFR-1 and anti-VEGFR-2 antibodies (Santa Cruz Biotech. Inc.)

The glioma cells were lysed using RIPA buffer with Protease and Phosphatase inhibitor (Beyotime). The total protein concentration was examined using a BCA kit. Equal amounts of total protein (30 μg) were electrophoresed in 10%–12% SDS‐PAGE and electro‐transferred onto NC membrane (GE). After blocking with 5% non‐fat dry milk in TBST for 1 h at room temperature (RT), the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with HRP‐labeled secondary antibodies (Proteintech) for 1 h at RT. An enhanced chemiluminescence reagent (Millipore) was used to detect protein expression value. The relative quantity of proteins was analyzed using the Image J software. The primary antibodies used are as follows: anti‐EZH2 (1:2000, #66476‐1‐Ig, Proteintech), anti‐VEGFA (1:1000, #66828‐1‐Ig, Proteintech), anti‐VEGFR2 (1:200, #sc‐6251, Santa‐Cruz Technology), anti‐Phospho^Tyr1175‐VEGFR2 (1:1000, #2478, CST), anti‐AKT (1:1000, #9272, CST), anti‐ Phospho^Thr308‐AKT (1:1000, #13038, CST), anti‐ERK1/2 (1:1000, #4695, CST), anti‐Phospho^{Thr202/Tyr204}‐ERK1/2 (1:1000, #4370, CST), anti‐GAPDH (1:5000, #60004‐1‐Ig, Proteintech), and anti‐β‐tubulin (1:5000, #10094‐1‐AP, Proteintech).