Figure 4.

Kinesin-derived angiogenesis inhibitor (KAI) inhibits vascular endothelial growth factor (VEGF)–induced endothelial cell (EC) migration. A: Schematic of domains of KIF13B and synthesized peptides used in the study. B: Pull-down assay for interaction of peptide KAI to vascular endothelial growth factor receptor 2 (VEGFR2), whereas CT23 control peptide did not interact with VEGFR2. C: VEGF-mediated migration of human primary umbilical vein endothelial cells (HUVECs) determined by wound healing scratch assay¹⁶ in the presence of 2.2 nmol/L VEGF with or without KAI (1 μmol/L). At indicated times, cells were fixed and stained with hematoxylin. D: Specificity of inhibitory effect of KAI determined by EC migration induced by different stimuli in Transwell migration assay as described.²⁷ HUVECs migrated toward 2.2 nmol/L VEGF, 50 ng/mL basic fibroblast growth factor (bFGF), or 1 μmol/L sphingosine-1-phosphate (S1P) were visualized by hematoxylin staining, and number of migrated cells were counted. Data are expressed as means ± SEM (C and D). n = 3 (C and D). ^∗P < 0.05 (one-way analysis of variance and Bonferroni multiple comparisons test). Scale bars = 200 μm (C and D). Cap-Gly, cytoskeleton-associated protein glycine-rich; FHA, forkhead associated; GST, glutathione S transferase; MBS, membrane-associated guanylate kinase–binding stalk; pro, proline rich; S, oligopeptide derived from RNase A (alias S-tag).