Figure 4.

(A and B) Angiostatin treatment leads to induction of kinase activity and tyrosine phosphorylation. BCE cells were cultured in the absence or presence of 2.5 μg/ml angiostatin in the growth medium for 24 h; during the last 10 min, cultures were treated with 100 ng/ml FGF-2 as indicated. Samples of cell extracts were analyzed by immunoblotting by using antiphosphotyrosine antibodies (A). Open arrow indicates a 140-kDa tyrosine-phosphorylated species; solid arrows indicate increased tyrosine phosphorylation in response to angiostatin. PAE cells were similarly treated and cell lysates were used for immunoprecipitation with antiphosphotyrosine antibodies, followed by in vitro kinase assays on the immobilized beads in the presence of [γ-³²P]ATP (B). The samples were separated by SDS/PAGE, and the gel was treated with 1 M KOH to reduce phosphorylation on Ser. Solid arrows indicate components with increased ³²P-labeled radioactivity in response to angiostatin; open arrow indicates a component that is not affected by angiostatin.