Anti vegf antibody

The protein expression levels of MDM2, p53, VEGF, caspase-3, cleaved caspase-3, and poly(ADP-ribose) polymerase (PARP) were analyzed by western blot. The following antibodies were used: anti-MDM2 antibody (1:1000; Sigma), anti-p53 antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-VEGF antibody (1:200; Santa Cruz Biotechnology), anti-caspase-3 antibody (1:1000; Santa Cruz Biotechnology), anti-cleaved caspase-3 p11 antibody (1:1000; Santa Cruz Biotechnology), and anti-PARP antibody (1:1000; Cell Signaling, Danvers, MA, USA).

Thirty μg of protein were loaded onto 10 % SDS gel coupled with loading buffer. Then the protein was transferred to nitrocellulose (NC) membrane and the nonspecific binding sites were blocked using 5 % non-fat dry milk. Then the NC membrane was incubated with diluted anti-ZNF217 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:200), anti-total CREB antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:200), anti-aromatase antibody (Abcam, Cambridge, UK) (1:1000), anti-VEGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (1:200) at 4 °C for overnight. After washing with TBST, membrane was incubated with diluted peroxidase-conjugated secondary antibodies for 1 h at room temperature. At last, the protein signals were detected using ECL western blotting substrate.

Gremlin, RAD001, rapamycin and perifosine were purchased from Sigma Chemicals (Shanghai, China). AZD8055 and OSI-027 were purchased from Selleck (Nanjing, China). All phosphorylation antibodies and their non-phosphorylated controls were obtained from Cell Signaling Tech (Danvers, MA). Anti-VEGF antibody and all other antibodies were purchased from Santa Cruz Biotech (Santa Cruz, CA).

The protein levels of VEGF and caspase-3 p17 in bone tissue were determined by western blot analysis. Bone tissue samples were lysed in RIPA buffer containing protease inhibitor cocktail (PIC, Sigma, USA). Lysate samples (50 µg protein) were electrophoresed on 15% SDS-PAGE gel and then transferred onto nitrocellulose membranes. The membranes were blocked and then incubated with anti-VEGF antibody (1 : 500, Santa Cruz Biotechnology, USA) and anti-caspase-3 antibody (1 : 1000, Santa Cruz Biotechnology, USA) overnight at +4°C followed by incubation with secondary antibodies: anti-rabbit IgG (H + L)-HRP conjugate (1 : 4000; Bio-Rad Laboratories, Inc., USA) and anti-mouse IgG (Fab specific)-Peroxidase (1 : 2500; Sigma, USA) for 1 h at room temperature. Thereafter, the membranes were developed with chemiluminescent agents: p-coumaric acid (Sigma, USA) and luminol (AppliChem GmbH, Germany). VEGF and caspase-3 tissue levels were normalized to β-actin (1 : 10000; Sigma, USA). The immunoreactive bands were quantified with Gel-Pro Analyzer v3.1 software.

Small intestine (100 mg) was lysed and homogenized in 1 ml lysis buffer (10 mM TBS, 1 mM EDTA, 1 mM EGTA, 2 mM sodium orthovanadate, 0.2 mM PMSF, 2 μg⁄ml leupeptin, 2 μg ⁄ml aprotinin, and 1% Triton X-100) for 30 min on ice and cleared by centrifugation at 12,000 g for 15 min at 4°C. Protein (80 μg) was fractionated on a 4 to 12% Bis-Tris gel and transferred to a 0.2-μm nitrocellulose membrane. Nitrocellulose blots were blocked by incubation in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1%Tween 20) containing 5% milk for 1 h at room temperature. Western blotting was performed using the following primary antibodies at 1:1,000 dilutions: anti-Bcl-xl antibody, anti-Bcl-2 polyclonal antibody (N-19), anti-Bax antibody, anti-VEGF antibody (Santa Cruz Biotechnology), and anti-pAkt antibody (Cell Signaling). After overnight incubation with the primary antibodies at 4˚C, the membranes were washed with TBST. Immunoreactive bands were detected using HRP-linked secondary antibody (Southern Biotech, Birmingham, AL) and the Enhanced Chemiluminescence (ECL) Western blot detection kit (Amersham, Piscataway, NJ). The immunoblots were exposed to X-ray film and analyzed with the NIH Image J analysis system. Mouse anti-β-actin monoclonal antibody (1:10,000; Sigma) was used as a loading control.