Effects of OSI-027 in Akt Thr308 phosphorylation and Akt-associated phosphatases. (A) Jeko (left) and Jurkat (right) cells were treated for 8 hours with diluent (lanes 1,8); OSI-127 at 1.25, 2.5, 5, 10 or 20μM (lanes 2-6 and 9-13, respectively); or 10nM rapamycin (lanes 7,14). Whole cell lysates28 were then subjected to SDS-PAGE followed by immunoblotting with antibody that recognizes the indicated antigen. (B) Jeko cells were treated for 24 hours with diluent (lanes 1,5); OSI-127 at 0.5, 2, or 5μM (lanes 2-4); or rapamycin at 1, 10 and 100nM (lanes 6-8). Cell lysates26 were then subjected to SDS-PAGE followed by immunoblotting with antibody that recognizes the indicated antigen. (C) After a 24-hour treatment with OSI-027, Akt immunoprecipitates (top) or cell lysates (bottom) from Jeko cells were probed with antibodies to PP1 and PP2A as indicated. (D, E) After a 24-hour treatment with OSI-027, immunoprecipitates of Akt2 (D) or Akt1 (E) from Jeko cells were probed for PHLPP isoforms as indicated. Additional experiments (not shown) indicated that PHLPP1 failed to immunoprecipitate with Akt1 and PHLPP2 failed to immunoprecipitate with Akt2.
