Figure 3.

Tyrosine phosphorylation of the adapter protein Shb and activation of mitogen-activated protein (MAP) kinases in response to FGF-2 occur in the presence of angiostatin. BCE cells in DME/10% newborn calf serum were cultured for 24 h with or without 2.5 μg/ml angiostatin, and in some cultures this was followed by 10-min stimulation with 100 ng/ml FGF-2. SDS-denatured cell lysates were immunoprecipitated by using affinity-purified anti-Shb antibodies, followed by SDS/PAGE, transfer to filter, and immunoblotting with antiphosphotyrosine antibodies (PY20, Upper). Immunoblotting of cell extracts separated by SDS/PAGE was performed, followed by transfer to filter and incubation with antibodies specifically recognizing activated MAP kinases Erk 1 and 2 (Lower).