IQGAP1-Dependent Signaling Pathway Regulates Endothelial Cell Proliferation and Angiogenesis
Figure 3
c-Src kinase activity is required for maximal phosphorylation of Y1173 of VEGFR-2.
Serum-starved PAE cells expressing wild type CKR, E1052/CKR and E1057/CKR were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes. Whole cell lysates (WCL) were subjected to Western blot analysis using an anti-phosphoY1173 specific-VEGFR-2 antibody (A), an anti-phosphoY1212 specific-VEGFR-2 antibody (B) or an anti-VEGFR-2 antibody (C). The graph represents the mean phosphorylation of Y1173 and Y1212 of VEGFR-2 (±SD) of three separate experiments. *P<0.01 (D). HEK-293 cells transiently expressing VEGFR-2 alone or co-expressing v-Src were stimulated with VEGF and immunoblotted with anti-pY1173 (E), anti-pY1052 (F), anti-pY1057 (G), anti-pY1212 (H), anti-c-Src (I) and anti-VEGFR-2 (J) antibodies. The blots were also quantified using Image J program (developed by NIH) and represents average tyrosine phosphorylation sites on VEGFR-2 of three separate experiments (±SD) from three separate experiments (K). SYF cells and SYF cells expressing chimeric VEGFR-2 (CKR) were either unstimulated (−) or stimulated with CSF-1 (+) for 10 minutes and cells lysates were prepared and immunoblotted with anti-pY1173 (L), anti-pY1052 (M) and anti-VEGFR-2 (N) antibodies. PAE cells expressing CKR were treated with different concentration of SU6656 for 30 minutes and stimulated with CSF-1 for 10 minutes and cell lysates were prepared as panel A and immunoblotted with anti-pY1173 (O), anti-pY1052 (P) and anti-VEGFR-2 (Q) antibodies. Phosphorylation of Y1173 and Y1052 in response to SU6656 treatment was quantified as panel D and is presented as the mean (±SD) of three separate independent experiments (R). A summary of the proposed model of phosphorylation Y1173 of VEGFR-2 by c-Src and VEGFR-2 itself is shown (S).