PAG formed a novel multiprotein complex with Fyn, Sam68, and RasGAP. (A) Increased Sam68 and RasGAP expression. Resting, anergic and rescued cells were untreated (−) or stimulated with MEM-92 for 2 minutes (+) before the lysis. Postnuclear lysates were blotted with specific antibodies against Sam68 and RasGAP. Actin staining is shown for equal loading. (B) To better determine whether Sam68 expression or localization was altered, cytoplasmic and nuclear fractions were prepared. GAPDH and Lamin A are presented as markers. (C) Sam68 and RasGAP associate with PAG. Cells were prepared as in panel A. PAG was then immunoprecipitated and associated proteins were detected by blotting with anti-RasGAP and anti-Sam68 antibody. The amount of precipitated material is shown with anti-PAG staining. (D) PAG, Fyn, and RasGAP association with Sam68. Samples prepared as in panel A were used to immunoprecipitate Sam68 and then probed with anti-RasGAP, anti-PAG, and anti-Fyn antibodies. The amount of precipitated material is shown by probing with anti-Sam68 antibody.