Transfection of the PH domain or the deletion mutant Gab2 molecules resulted in a compromised activation of PI3 but not Erk kinases.
Wild-type, mutant Gab2, and control vector-transfected Ba/F3 cells were starved and stimulated by β1-integrin cross-linking for 5 minutes. (A) Cell lysates were prepared and immunoprecipitated using anti-Gab2 Ab was performed. Immunoprecipitates were resolved by SDS-PAGE and blotted with antiphosphorylated tyrosine, anti-SHP-2, anti-p85, and anti-Gab2 Abs. Serum control immunoprecipitation (c) was performed using the cell lysates of stimulated wild-type Gab2-transfected cells. (B) Cell lysates were immunoprecipitated using anti-p85 subunit of PI3 kinase Ab. Immunoprecipitates were washed and subjected to PI3 kinase assay as described in detail in “Materials and methods.” 32P-labeled phospholipids were visualized by autoradiography. Shown in the lower panel is the loading control of PI3 kinase precipitated by anti-p85 Ab. Serum control immunoprecipitation was performed using the cell lysates of stimulated wild-type Gab2 transfected cells. (C) Whole cell lysates (20 μg) were resolved by SDS-PAGE and blotted with antiphospho Erk Ab. For the loading control, membranes were stripped and reblotted with anti-Erk Ab. The pictures shown are representative of 2 independent experiments.
