Figure 3.

Assembly of the Grb2-PI3KC2β-Eps8-Abi1-Sos multiprotein complex. (A) Grb2 was immunoprecipitated from Triton X-100–insoluble A-431-C2β WT cell lysates and probed with the antibodies to Sos1, PI3KC2β, Eps8, and Grb2. (B) Abi1 interacts with Grb2. Lysates from A-431 and A-431-C2β WT cell line were immunoprecipitated with either the N- or C-terminal SH3 Gst-Grb2 fusion proteins. Precipitated proteins from the respective lysates were immunoblotted with Abi1 and Eps8. (C) Abi1 and control rabbit Immune-globulin complexes from Triton X-100–insoluble lysates of A-431 cells were Western-blotted with anti-Abi1 and anti-Grb2 antibodies. (D) A-431-C2β WT cells were serum-starved for 24 h (−) and stimulated with EGF for 5 min (+). Cell lysates were immunoprecipitated with antibodies to Myc, Grb2, Eps8, Abi1, or Sos1 then Western blotted with antibodies to Abi1. (E) Immune-complex class II PI3K lipid kinase activity was evaluated in the presence of calcium ions as previously described (Arcaro et al., 1998). The results represent the mean of two independent experiments.