Activation of ErbB receptors by EGF-like motifs of human proEGF. (A) GST fusion proteins containing EGF-like motifs 1–4, 5–8, or 5–9 of the EGF precursor were immobilized on glutathione-agarose beads. For control, GST fusion proteins containing EGF or NDF were used. The beads were incubated for 1 h at 4°C with conditioned media containing 1 μg of the indicated IgB protein. Protein complexes were immunoblotted with an anti-human Fc antiserum for detection of bound IgBs. (B) Monolayers of the indicated human breast cancer cell lines were incubated, for 10 min at 37°C, in the presence of 100 ng/ml GST fusion proteins or 5 ng/ml ligands (EGF or NDF). Receptor activation was detected by an antiphosphotyrosine antibody.