(A) Proteins associated with Grb2/Ash in vitro and their tyrosine phosphorylation. The lysates from UT-7 cells unstimulated (lane 2) or stimulated with GM-CSF (lane 1) were mixed with the GST fusion protein including the full-length Grb2/Ash noncovalently coupled to glutathione-agarose beads. Beads were resuspended in Laemmli's sample buffer, subjected to SDS-PAGE, and immunoblotted with anti-Py (4G10). Molecular weight markers, indicated at the left, are given in kilodaltons. The arrows indicate the position of pp52, pp66, pp130, and pp135. (B) In vitro association of pp130 and pp135 with the SH3 domain of Grb2/Ash and tyrosine phosphorylation of pp130 and pp135. The lysates from UT-7 cells unstimulated (lanes 1 and 3) or stimulated with GM-CSF (lanes 2 and 4) were mixed with GST fusion protein including the SH2 domain (lanes 1 and 2) or the N-terminal SH3 domain (lanes 3 and 4) of Grb2/Ash noncovalently coupled to glutathione-agarose beads. The resulting precipitates were resuspended in Laemmli's sample buffer, subjected to SDS-PAGE, and immunoblotted with anti-Py (4G10). Molecular weight markers, indicated at the left, are given in kilodaltons. The arrows indicate the position of pp52, pp66, pp130, and pp135.