Proteome profiler human angiogenesis antibody array kit

Cell culture medium (RPMI-1640, DMEM) and serum (fetal bovine serum (FBS)) were purchased from Invitrogen (Gaithersburg, MD, USA). Simvastatin, atorvastatin, lovastatin, and pravastatin were purchased from Sigma-Aldrich (St Louis, MO, USA). Calcein-AM was from BD Biosciences (San Jose, CA, USA), and generic chemicals were purchased from Sigma-Aldrich. The Proteome Profiler Human Angiogenesis Antibody Array Kit was purchased from R&D Systems (Minneapolis, MN, USA). Angiopoietin 2 siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Binding immunoglobulin protein (BiP) and Hsp90α siRNAs were purchased from Qiagen (Valencia, CA, USA). Angiopoietin 2, BiP, and Hsp90α active protein were purchased from Abcam (Cambridge, UK). The rabbit polyclonal anti-angiopoietin 2 antibody was purchased from Abfrontier (Seoul, Korea), mouse monoclonal anti-BiP antibody from BD Biosciences (San Jose, CA, USA), rat monoclonal anti-Hsp90α antibody from Stressgen (Victoria, BC, Canada), mouse monoclonal anti-CD31 antibody from Dako (Glostrup, Denmark), and mouse monoclonal anti-BrdU antibody from Roche (Mannheim, Germany).

Lenvatinib was purchased from AdooQ Bioscience LLC, and a solution of Lenvatinib was prepared by dilution with DMSO and stored at -20˚C. RPMI-1640 was obtained from Gibco (Thermo Fisher Scientific, Inc.). Trypan blue was purchased from MilliporeSigma. DMEM, minimum essential medium (MEM), and FBS were obtained from Wako Pure Chemical Industries, Ltd. Penicillin-streptomycin was obtained from Invitrogen (Thermo Fisher Scientific, Inc.). A cell cycle phase determination kit was obtained from Cayman Chemical Company, a protease inhibitor cocktail from iNtRON Biotechnology, and a Proteome Profiler Human Angiogenesis Antibody Array Kit from R&D Systems, Inc.

To study the mechanism of angiogenesis inhibited by simvastatin, the Proteome Profiler Human Angiogenesis Antibody Array Kit (R&D Systems) was used according to the manufacturer's instructions. The Human Angiogenesis Antibody Array Kit detects 55 different angiogenesis-related proteins (activin A, ADAMTS-1, angiogenin, angiopoietin1, angiopoietin2, angiostatin, amphiregulin, artemin, coagulation factor III, CXCL16, DPPIV, EGF, EG-VEGF, endoglin, endostatin, endothelin-1, FGF acidic, FGF basic, FGF-4, FGF-7, GDNF, GM-CSF, HB-EGF, HGF, IGFBP-1, IGFBP-2, IGFBP-3, IL-1β, IL-8, LAP, leptin, MCP-1, MIP-1α, MMP-8, MMP-9, NRG1-β1, PTX3, PD-ECGF, PDGF-AA, PDGF-AB, persephin, PF4, prolactin, serpin B5, serpin E1, serpin F1, TIMP-1, TIMP-4, thrombospondin-1, thrombospondin-2, uPA, vasohibin, VEGF, and VEGF-c). Briefly, membranes were blocked with array buffer 7 and then incubated with colon cancer conditioned media of CoLo320, NCI-H716, and SW48 cells overnight at 4 °C. The membranes were washed three times with 1 × wash buffer and incubated with the streptavidin-HRP (1 : 5000 dilution) for 30 min. After three washes of 10 min each, the membranes were incubated with the ECL Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and exposed to X-ray film.