Recombinant human vegf vegf165

Hesperetin (Fig. 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The compound was dissolved in 100% dimethyl sulfoxide (DMSO). A 100 mmol/L stock solution of hesperetin was prepared and stored as small aliquots at −20°C until needed. We purchased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMSO, gelatin, and horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit antibodies from Sigma-Aldrich. Recombinant human VEGF (VEGF₁₆₅) was obtained from R&D Systems (Minneapolis, MN, USA). Growth factor-reduced Matrigel was purchased from BD Biosciences (San Jose, CA, USA). The antibodies p-p38 (Thr¹⁸⁰/Tyr¹⁸²), p-JNK (Thr¹⁸³/Tyr¹⁸⁵), JNK, p-PI3K (Tyr⁴⁵⁸), PI3K and p-AKT (Ser⁴⁷³), AKT were purchased from Cell Signaling Technology (Danvers, MA, USA). The HRP-conjugated β-actin, ERK, p38α and p-ERK (Thr²⁰²/Tyr²⁰⁴) antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

Limonin (purity >99.37%) was purchased from Shanghai Winherb Medical Science. A 50 mmol/L stock solution was prepared in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO), Regorafenib were purchased from Selleck Chemicals (Houston, TX). Growth factor-reduced Matrigel was purchased from BD Biosciences (San Diego, CA). Primary antibodies against phosphor-specific anti-VEGFR2 (Tyr¹¹⁷⁵), VEGFR2, phospho-STAT3 (Tyr⁷⁰⁵), STAT3, Bcl2, BCL-xl, MMP-9 and SHP-1 were obtained from Cell Signaling Technology (Danvers, MA, USA; 1:1,000 dilution). VEGF (A-20), IGFR, phospho-IGFR(Tyr¹³¹⁶) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA; 1:1,000 dilution). β-actin and GAPDH were obtained from Hangzhou Huaan Biotechnology Company(China; 1:1,000 dilution). Goat Anti-Rabbit IgG and HRP Goat Anti-Mouse IgG were provided from Abbkine (1:2,500 dilution). Anti-CD31 and anti-Ki67 were provided by Abcam (UK; 1:200 dilution). SiRNA and scrambled control was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human VEGF (VEGF₁₆₅) was acquired from R&D Systems (MN, USA). All other reagents were obtained from Sigma-Aldrich (St Louis, MO, USA).