GAB1 association with PI 3-kinase in Epo-stimulated cells. (A) Anti–PI 3-kinase immunoprecipitates were prepared from Epo-stimulated UT-7 cells as described in Fig 1. Half of the immunoprecipitates were saved for direct analysis by Western blot and the remaining was denatured by boiling in nonreducing Laemmli sample buffer, diluted with solubilization buffer, and reimmunoprecipitated using anti-GAB1 antibodies. Both samples were analyzed by Western blot using antiphosphotyrosine (anti-PY) antibodies. (B) UT-7 cells were serum- and growth factor-starved for 18 hours and incubated for 10 minutes in the presence (+) or absence (−) of 10 U/mL Epo. The cells were then lysed using 1% Brij 98 and cleared lysates from 10⁷ cells were immunoprecipitated using anti-GAB1 antibodies. Immunoprecipitates were analyzed by Western blot using antiphosphotyrosine antibodies (PY). The blot was stripped and reprobed with anti–PI 3-kinase antibodies and anti-GAB1 antibodies, successively. (C) Anti-GAB1 immunoprecipitates from UT-7 cells stimulated for 10 minutes with 10 U/mL Epo or from control cells were prepared and tested for PI 3-kinase activity as described in Materials and Methods. The lipid products were separated by thin-layer chromatography, and the migration position of phosphatidylinositol 3-phosphate (PIP) was determined by comparison with authentic unlabeled PIP run in adjacent lanes and shown by iodine staining. “Ori” indicates the origin of migration.