The association between Shc and p140 is mediated through the PTB domain of Shc and this complex is constitutively activated in some AML. (A) hGM-CSF and CSF-1 induce the association of tyrosine-phosphorylated p140 with the PTB domain of Shc. Cell lysates from unstimulated (lanes 1, 3, and 5) or hGM-CSF–stimulated (lanes 2, 4, and 6) TF-1 cells or from unstimulated (lane 7) or CSF-1-stimulated (lane 8) BAC1.2F5 cells were immunoprecipitated with anti-Shc antibodies (lanes 1 and 2) or adsorbed with either MBP vector (lanes 5 and 6) or MBP fusion proteins containing the N-terminal PTB domain of Shc (MBP-N-Shc; lanes 3, 4, 7, and 8). Associated tyrosine-phosphorylated proteins were analyzed by antiphosphotyrosine immunoblotting. (B) p140 is constitutively associated with the PTB domain of Shc in AML. Cell lysates of unstimulated primary AML were either incubated with MBP vector protein (AML-3; lane 1) or MBP-N-Shc fusion protein (AML 3 through AML-7; lanes 2 through 6), and the adsorbed proteins were analyzed by antiphosphotyrosine immunoblotting. The AML samples analyzed in this experiment correspond to the AML-3 through AML-7 samples analyzed in Fig 4B, lanes 2 through 6.