The pp135 constitutively binds to the SH3 domain of Grb2/Ash and inducibly becomes tyrosine-phosphorylated with the stimulation of GM-CSF. The lysates from UT-7 cells stimulated with GM-CSF (lanes 2 and 4) or unstimulated (lanes 1 and 3) were mixed with GST-Grb2/Ash SH3 (lanes 1 and 2) or with anti-P6 (lanes 3 and 4). The resulting precipitates were subjected to SDS-PAGE and immunoblotted with anti-Py (A) and reprobed with anti-P6 (B). The arrow indicates the position of pp135. Molecular weight markers, indicated at the left, are given in kilodaltons. (C) In vivo association of pp135 with Grb2/Ash. The lysates from UT-7 cells stimulated with GM-CSF (lane 2) or unstimulated (lane 1) were mixed with anti-P6. The resulting precipitates were subjected to SDS-PAGE and immunoblotted with anti-Grb2/Ash. The total UT-7 cell lysate (TCL, lane 3) is also applied for reference. Molecular weight markers, indicated at the left, are given in kilodaltons. The arrow indicates the position of Grb2/Ash.