Fig. 1. Biochemical analysis of endogenous ErbB family members in SKBR3 cells. (A) Tyrosine phosphorylation status of ErbB receptors in SKBR3 cells after 2 hours serum starvation (0) or after indicated time course of treatment with either EGF (20 nM) or heregulin (3.2 nM). (B) Kinase activity associated with EGFR or ErbB2 immunoprecipitates prepared from resting, EGF- or heregulin-treated cells, measured in vitro by K-LISA. (C) Effects of kinase inhibitors on EGFR or ErbB2 kinase activity, measured by K-LISA in immunoprecipitates prepared from cell lysates after a 2-minute stimulus with 20 nM EGF. K-LISA values are corrected for baseline color development typical in IgG controls. (D) Co-precipitation assay for ErbB heterodimerization. Where indicated, cells were stimulated with 20 nM EGF or 3.2 nM heregulin, followed by lysis with 1% NP-40 and immunoprecipitation using EGRF-, ErbB2- or ErbB3-specific antibodies. Samples were subjected to SDS-PAGE, followed by immunoblotting with ErbB-specific antibodies. (E). Immunoblotting of equal amount of SKBR3 lysates (10 µg of protein each lane) to document the relative levels of all three ErbB3 family members.