Figure 4
Effects of anosmin-1 deletion mutants on VEGFR2 activation and cell migration. (a) Expression of VEGFR2 in PAE and PAE/KDR cells. Cell lysates were analyzed by western blotting with the anti-VEGFR2 and anti-β-actin Abs. (b) Transwell cell migration assay. PAE or PAE/KDR cells were seeded in the upper compartment, and were treated with the indicated concentrations of anosmin-1 or VEGF-A, or without anosmin-1 (Control). The cells that moved into the lower chamber were counted in five different microscopic fields. **P < 0.01 vs. PAE; ††P < 0.01 vs. Control. (c) Schematic structures of anosmin-1 deletion mutants. WAP: whey acidic protein-like domain. (d) Generation of recombinant proteins of anosmin-1 WT and mutants. The purified recombinant proteins (0.5 μg each) were used for SDS-PAGE and visualized by SyproRuby staining. (e) Transwell cell migration assay. PAE/KDR cells (5.0 × 104 cells/well) were seeded in the upper compartment, and were stimulated with 7.5 nM anosmin-1 WT or the indicated deletion mutants, or without anosmin-1 (Control). The cells that moved into the lower chamber were counted in five different microscopic fields. **P < 0.01 vs. WT; ††P < 0.01 vs. Control. (f) Phosphorylation of VEGFR2 by treatment with anosmin-1 deletion mutants. Starved PAE/KDR cells were treated with 7.5 nM anosmin-1 WT or the indicated deletion mutants for 5 min or with 1 nM VEGF-A for 2 min. As a negative control (Control), cells were incubated without reagents. 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.
