The C-terminal SH3 domain of Grb2 binds to tyrosine-phosphorylated p140 in AML. (A) p140 is constitutively associated with Grb2 in AML cells. Cell lysates from unstimulated (lane 1) or hGM-CSF–stimulated TF-1 cells (lane 2) and from unstimulated primary leukemia cells obtained from five patients with AML (AML-3 through AML-8; lanes 3 through 8) were immunoprecipitated with anti-Grb2 antibodies followed by antiphosphotyrosine immunoblotting (P.Tyr). The position of p140 is indicated on the left. (B) The C-terminal SH3 domain of Grb2 binds to p140 in AML cells. Cell lysates from unstimulated primary AML cells were incubated with either GST vector protein (GST; AML-3; lane 1) or with GST fusion proteins containing the C-terminal SH3 domain of Grb2 (GST-Grb2-SH3; AML-3 through AML-8; lanes 2 through 7). Associated tyrosine-phosphorylated proteins were analyzed by antiphosphotyrosine immunoblotting (lanes 1 through 7). The AML samples analyzed in this experiment correspond to the AML-3 through AML-8 samples analyzed in (A), lanes 3 through 8, and in Fig 4B, lanes 2 through 7. The position of tyrosine-phosphorylated proteins is indicated on the left. Molecular weight markers are indicated on the right.