Figure 3
Anosmin-1-induced activation of VEGFR2 for tube formation. (a) Phosphorylation of VEGFR2 by anosmin-1 treatment. Starved HUVECs were stimulated with 7.5 nM anosmin-1 for the indicated durations or with 1 nM VEGF-A for 2 min. Cell lysates were analyzed by western blotting with the indicated Abs. The density of each phosphorylated VEGFR2 band normalized by the VEGFR2 band was measured, and the density relative to the value without treatment (0 min) set as 1.0 was calculated and is shown below the band. (b) Suppression of anosmin-1-induced tube formation by VEGFR2 inhibitor. HUVECs were seeded onto a Matrigel-coated 24-well plate, and then, 7.5 nM anosmin-1 or 1 nM VEGF-A was added with the VEGFR2 inhibitor (10 μM SU5614) or 0.1% DMSO at the same time. As a negative control (Control), cells were incubated without reagents in the presence of the VEGFR2 inhibitor or 0.1% DMSO. After the incubation and fixation, the formed tubes were observed by light microscopy. Scale bar: 500 μm. (c) Summary graphs of (b). Total length of the formed tubes was measured. **P < 0.01 vs. DMSO; ††P < 0.01 vs. Control.
