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Fig. 4 Effect of WA-25 on VEGFR2 expression in HUVECs. HUVECs were treated with WA-25 (1–20 µM) for 48 h and separately subjected to VEGFR2 mRNA and protein expression assay. (A) VEGFR2 mRNA level was determined using quantitative qRT-PCR analysis. (B) Dose-dependent (48 h) and (C) time-dependent effect of WA-25 on VEGFR2 protein expression was measured using Western blot analysis (D) Flow cytometric analysis of surface VEGFR2 expression after WA-25 (20 µM) treatment for 48 h (E) VEGF and VEGFR2 protein expressions were analyzed using immunofluorescence. After WA-25 (20 µM) treatment for 48 h, the cell surface VEGFR2 expression in endothelial cells was analyzed using FACScan. Data are represented as mean ± SD in triplicates. * p < 0.05; ** p < 0.01, versus control groups.