Vegf165

Blank ELISA plates were coated with VEGFR2-his (Sino Biuological Inc., Beijing, China, #HPLC-10012-H08H) and blocked with 1% BSA in PBS. Different concentrations of BD0801 (Jiangsu Simcere Pharmaceutical Co., Ltd., Nanjing, China) or bevacizumab (Genentech/Roche, San Francisco, CA, USA) were incubated with human VEGF₁₆₅ (PrimeGene, Shanghai, China, #105–05) at 37 °C for 1 h before adding into the plates coated with VEGFR2-his for incubation. Unbounded VEGF₁₆₅ was washed off, and the primary antibody anti-VEGF rabbit mAb (Sino Biological, #11066-R105) was added for incubation. After washing the plates, the secondary antibody Peroxidase AffiniPure Donkey Anti-Rabbit IgG Jackson (Jackson ImmunoResearch, West Grove, PA, USA, #711–035-152) was added. After incubation, the redundant HRP complex was washed off. Finally, peroxidase (HRP) substrate TMB (Thermo Fisher Scientific, Waltham, MA, USA, #34029) was added, and the OD value was measured by SpectraMax I3X (Molecular Devices, LLC, Sunnyvale, CA, USA). The detection wavelength was 450 nm, and the reference wavelength was 630 nm. The IC₅₀ was calculated based on the OD value at different concentrations by GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Each concentration was measured in duplicates in each experiment, and the experiment was repeated three times.

2 × 10⁴ HUVECs were seeded into the 96-well plate coated with matrigel (Corning, Bedford, USA), and added with the supernatants obtained from SHH002-hu1 treated MDA-MB-231/MDA-MB-468 cells. After 8-h incubation, endothelial tube formation was photographed with an inverted OLYMPUS microscope, 10 ng/mL VEGF₁₆₅ (Sino Biological, Beijing, China) treated HUVECs were as control. The endothelial tubes were counted with Image-Pro-Plus program and the tube formation rate quantified on the basis of the control.