Angiostatin inhibits phosphorylation of c-met by HGF in HUVECs.

HUVECs were cultured in serum-free medium as described in “Materials and methods,” then stimulated with HGF (10 ng/mL; panel A) or with VEGF (10 ng/mL; panel B) for 5 minutes prior to preparation of cell lysates. The lysates were separated by SDS-PAGE and subjected to immunoblotting. (A) Immunoblotting for Phospho–c-met: lane 1 indicates control cells; lane 2, cell lysates after stimulation with HGF (10 ng/mL); lane 3, cells treated with HGF and angiostatin (1 μM); and lane 4, cells treated with HGF and angiostatin (3 μM). (B) Immunoblotting for Phospho-KDR: lane 1 indicates control cells; lane 2, cell lysates after stimulation with VEGF (10 ng/mL); lane 3, cells treated with VEGF and angiostatin (1 μM); and lane 4, cells treated with VEGF and angiostatin (3 μM). Angiostatin inhibited the phosphorylation of c-met induced by HGF but did not inhibit KDR phosphorylation induced by VEGF. Blots were stripped and re-probed for actin as a loading control.