FIG. 6.
Effect of mutating A84 on the behavior of 4E-BP3. Mutants were created in which A84 in 4E-BP3 was replaced by serine (A84S, panel A) or glutamate (A84E, panel B). These variants were expressed in HEK293 cells in parallel with the wild-type protein. Cells were starved of serum overnight and in some cases were treated for 60 min with 100 nM insulin. Extracts were subjected to affinity chromatography on m7GTP-Sepharose followed by analysis of the bound material by SDS-PAGE and immunoblotting with the indicated antibodies.