Anti vegf antibody

The whole-cell protein extracts were prepared by lysing the cells with 2% CHAPS lysis buffer in the presence of 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 5 mM EDTA and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA). The extracted proteins were resolved on a 4-12% polyacrylamide SDS-PAGE, as previously described (23 (link),24 (link)). The primary antibodies used were as follows: anti-RET (#3220), anti-RET/PTC1 (#14698), anti-cMYC (#5605), anti-p-mTOR (#2971) and anti-mTOR (#2972) (dilution 1:1,000) were purchased from Cell Signaling Technology (Beverly, MA, USA), anti-Bcl-2 (sc-7382), anti-pERK (sc-7383), anti-ERK (sc-271270), anti-cyclin D1 (sc-20044) and anti-β-actin (sc-47778) (dilution 1:300) were purchased from Santa Cruz Biotechnology. Anti-VEGF antibody was purchased from GeneTex (Irvine, CA, USA; #GTX102643). Mouse and rabbit IgG antibodies tagged with horseradish peroxidase (HRP) (Bio-Rad Laboratories, Hercules, CA, USA) were used as secondary antibodies (dilution 1:1,000). An enhanced chemiluminescence substrate kit (#32106) purchased from Thermo Fischer Scientific was used for detection.

Cells were lysed and extracts were prepared as described previously 22 . HIF-1α, HIF-2α, FOXC1, VEGF, E-cadherin and Fibronectin proteins in human cells were detected in 150 µg of cell extract using monoclonal anti-HIF-1α antibody (diluted 1:650; Novus), anti-HIF-2α antibody (diluted 1:700; Cell Signaling Technology), anti-FOXC1 antibody (1:1000; Novus), anti-VEGF antibody (1:2000; Genetex), E-cadherin antibody (diluted 1:1000; Novus) and Fibronectin antibody (diluted 1:1000; Novus). Western blots were normalized using a monoclonal anti-β-actin antibody (diluted 1:10,000; Sigma-Aldrich).