Angiostatin binds to soluble c-met.

(A) Media containing the soluble, truncated c-met receptor derived from A431 cells were adsorbed to an EIA/RIA plate. Angiostatin was added in concentrations of 0 to 5 μM and bound angiostatin was detected with a polyclonal antibody to plasminogen followed by an alkaline phosphatase–conjugated anti–rabbit IgG (solid line). Antibody detection was quantified using p-nitrophenylphosphate and measuring the V_max at OD₄₀₅. At each concentration of angiostatin, Voller buffer was used to show nonspecific binding of angiostatin to the plate, which was subtracted from total binding to obtain specific binding (displayed in this graph). Replicates of n = 3 were performed. The error bars represent ± 1 SD. (B) Coimmunoprecipitation. A431 medium containing soluble c-met was incubated with angiostatin, then immunoprecipitated with nonimmune mouse IgG (lane 1) or with anti–c-met extracellular domain (lane 2). Immunoblotting was performed with antiplasminogen. Lane 3: angiostatin (positive control). Angiostatin was immunoprecipitated by anti–c-met, demonstrating a soluble c-met/angiostatin complex.