American Society of Hematology

Fig. 3.

$Fig. 3. Gab2 associates with SHP-2 and PI-3 kinase. (A) BAF-B03 cells were stimulated with IL-3 for the indicated periods of time. Proteins were immunoprecipitated with an anti-Gab2 antibody, separated on an SDS-polyacrylamide gel, and subjected to immunoblotting with anti-phosphotyrosine, anti-Gab2, anti-SHP-2, and anti-p85 PI-3 kinase antibodies. The locations of Gab2, SHP-2, and p85 are indicated by arrows. (B) BAF-B03 cells were stimulated with IL-3 (+) or left unstimulated (−). Cell lysates were immunoprecipitated with anti–SHP-2 or anti-p85 PI-3 kinase antibodies and subjected to immunoblotting with anti-phosphotyrosine (upper panel) and anti-Gab2 (lower panel) antibodies. (C) 293T cells were transfected with an expression vector for Flag-tagged Gab2 (lane 1) or a control vector (lane 2), and cell lysates were immunoprecipitated with anti-Flag antibodies. Cell lysates from IL-3–stimulated BAF-B03 cells were immunoprecipitated with anti-SHP-2 (lane 3) or anti-Gab2 (lane 4) antibodies. The immunoprecipitates were separated on the same SDS-polyacrylamide gel. The anti-Flag, anti-SHP-2, and anti-Gab2 immunoprecipitates were analyzed by immunoblotting with anti-Flag and anti-phosphotyrosine antibodies, respectively. (D) Cell lysates from BAF-B03 cells that were stimulated or unstimulated with IL-3 were mixed with GST fusion proteins containing the entire coding fragment (Full) or the SH2 domain of Grb2. GST fusion protein-bound fractions were isolated by glutathione sepharose and subjected to immunoblotting with anti-phosphotyrosine (upper panel) and anti-Gab2 (lower panel) antibodies. Anti-SHP-2 immunoprecipitates were also analyzed in the same membrane. Note that the slower migrating form of Gab2 is the tyrosine-phosphorylated form. (E) BAF-B03 cells expressing the chimeric receptor G277 (containing the entire cytoplasmic domain) or G68 (containing 68 amino acids from the membrane region) were stimulated with G-CSF (+) or left unstimulated (−), and immunoprecipitated Gab2 proteins were subjected to immunoblotting with anti-phosphotyrosine (upper panel) and anti-Gab2 (lower panel) antibodies.$

Gab2 associates with SHP-2 and PI-3 kinase. (A) BAF-B03 cells were stimulated with IL-3 for the indicated periods of time. Proteins were immunoprecipitated with an anti-Gab2 antibody, separated on an SDS-polyacrylamide gel, and subjected to immunoblotting with anti-phosphotyrosine, anti-Gab2, anti-SHP-2, and anti-p85 PI-3 kinase antibodies. The locations of Gab2, SHP-2, and p85 are indicated by arrows. (B) BAF-B03 cells were stimulated with IL-3 (+) or left unstimulated (−). Cell lysates were immunoprecipitated with anti–SHP-2 or anti-p85 PI-3 kinase antibodies and subjected to immunoblotting with anti-phosphotyrosine (upper panel) and anti-Gab2 (lower panel) antibodies. (C) 293T cells were transfected with an expression vector for Flag-tagged Gab2 (lane 1) or a control vector (lane 2), and cell lysates were immunoprecipitated with anti-Flag antibodies. Cell lysates from IL-3–stimulated BAF-B03 cells were immunoprecipitated with anti-SHP-2 (lane 3) or anti-Gab2 (lane 4) antibodies. The immunoprecipitates were separated on the same SDS-polyacrylamide gel. The anti-Flag, anti-SHP-2, and anti-Gab2 immunoprecipitates were analyzed by immunoblotting with anti-Flag and anti-phosphotyrosine antibodies, respectively. (D) Cell lysates from BAF-B03 cells that were stimulated or unstimulated with IL-3 were mixed with GST fusion proteins containing the entire coding fragment (Full) or the SH2 domain of Grb2. GST fusion protein-bound fractions were isolated by glutathione sepharose and subjected to immunoblotting with anti-phosphotyrosine (upper panel) and anti-Gab2 (lower panel) antibodies. Anti-SHP-2 immunoprecipitates were also analyzed in the same membrane. Note that the slower migrating form of Gab2 is the tyrosine-phosphorylated form. (E) BAF-B03 cells expressing the chimeric receptor G277 (containing the entire cytoplasmic domain) or G68 (containing 68 amino acids from the membrane region) were stimulated with G-CSF (+) or left unstimulated (−), and immunoprecipitated Gab2 proteins were subjected to immunoblotting with anti-phosphotyrosine (upper panel) and anti-Gab2 (lower panel) antibodies.

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