Gab2 is involved in the activation of ERK MAP kinase. (A) 293T cells expressing the chimeric receptor G277 were transfected with expression vectors for Flag-tagged ERK2, together with either Gab1, Gab2, or a control vector. Cells were stimulated with G-CSF for 30 minutes (+) or left stimulated (−) and the ERK2 activities were determined by an immunoprecipitation kinase assay using myelin basic protein (MBP) as a substrate. The amount of MBP-incorporated³²P was quantified by an image analyzer and indicated as the ratios against that from control unstimulated cells. (B) 293T cells were transfected with vectors for Flag-tagged ERK2, together with either Gab1, Gab2, or a control vector. They were also transfected with the expression vectors for v-Src (+) or a control vector (−), as indicated. The ERK2 activities were determined as described above. (C) 293T cells were transfected with vectors for Flag-tagged-Gab1 or Gab2, together with either JAK1, v-Src, Btk, Tec, or a control vector. Gab1 and Gab2 were immunoprecipitated with anti-Flag antibodies and analyzed using anti-phosphotyrosine (upper panel) and anti-Flag antibodies (lower panel).