Figure 4
Angiostatin inhibits phosphorylation of c-Met and Akt/PKB in HMEC-1 cells. HMEC-1 cells were incubated at 37°C and 5% CO2 for 40 h. For the treatment with plaminogen mixture, 2 μg/well recombinant MMP-19, or aprotinin 1 ug/ml were used. Cell lysates were prepared and analyzed by 12.5% SDS-PAGE followed by immunoblotting for phospho-cMet (A) and phospho-Akt/PKB (B). Blots were then stripped and re-probed for β-actin as a loading control. Quantification of phospho-cMet and phospho-Akt/PKB protein was performed by densiometric analysis using the Aida Image Analyser and normalized for the signal intensity of the beta-actin band. Densitometric analysis; intensity of phospho-c-Met (B) and phospho-Akt/PKB (D) bands was normalized to the corresponding signal intensity of β-actin bands and the buffer control was set as 100%. Sodium phosphate, used as buffer for the above experiments, was used as a control. One of two independent experiments is shown.