Figure 5.

 Phosphorylation of 4E-BP1 by Akt inhibits interaction with eIF4E. Rat1a and Rat1a/MyrAkt cells were incubated in 0.5% FCS overnight. Rat1a cells were then treated with 20% FCS for 40 min. Cells were lysed by freeze–thaw cycles and extracts were either heat treated (total extract; 100 μg) or incubated (750 μg) with m⁷GDP–agarose resin, as described in Materials and Methods. Samples were separated by SDS-PAGE and 4E-BP1 protein was analyzed by Western blotting. (A) Total extract (100 μg). (B) Material bound to the m⁷GDP–agarose resin. Positions of the 4E-BP1 isoforms are indicated.