Figure 4.

 The phosphopeptide map of 4E-BP1 in 293 MyrAkt cells is identical to that of serum-stimulated 293 cells. ³²P-Labeled 4E-BP1 (Fig. 3) was excised from an Immobilon membrane, digested with trypsin–chymotrypsin, and analyzed by two-dimensional phosphopeptide mapping, as described in Materials and Methods. HEK 293 cells (A–D) and HEK 293/ MyrAkt cells (E–G) were deprived of serum for 36 hr. Cells were labeled with ³²P as described in Materials and Methods. (A,E) Untreated cells (B–D,F,G) were treated as follows: with 15% FCS for 30 min (B); pretreated with rapamycin (20 ng/ml) for 20 min before addition of FCS (C); pretreated with wortmannin (100 nm) for 20 min before addition of FCS (D); with rapamycin (20 ng/ml) for 20 min (F); with wortmannin (100 nm) for 20 min (G).