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Fig. 9
QODG inhibits VEGF-induced phosphorylation of VEGFR2 kinase and VEGFR2-mediated signaling pathway downstream molecules in HUVECs.
(A) QODG inhibited VEGF-induced phosphorylation of VEGFR2 in a dose-dependent manner, but phospho-VEGFR1 protein, the total amount of VEGFR1 and VEGFR2 proteins in each sample of cells all remained comparable. After probed with the antibodies anti-VEGFR2, anti-VEGFR1, anti-phospho-VEGFR2 and anti-phospho-VEGFR1, total VEGFR2 and VEGFR1 proteins, and phospho-VEGFR2 and VEGFR1 proteins in different groups were examined by Western blotting analysis. Three independent experiments were performed in triplicates. (B) QODG inhibited VEGFR2 kinase activity. Inhibition of VEGFR2 kinase activity by QODG was analyzed using an in vitro HTScan® VEGF receptor 2 kinase kit (Cell Signaling Technology, Danvers, MA, USA) combined with colorimetric ELISA detection according to the manufacturer′s instructions. The reaction processed with only DMSO (0.1%) served as a vehicle control. Data are expressed as percentages of the vehicle control. Three independent experiments were performed. (C, D) QODG inhibited the activation of VEGFR2-mediated downstream signaling. The activation of c-Src, FAK and ERK (C), AKT, mTOR and p70S6K (D) was suppressed by QODG. After probed with specific antibodies, proteins in different groups were examined by Western blotting analysis. Three independent experiments were performed in triplicates.