FIG. 5.

In vivo interaction of Eap1p with eIF4E is dependent on the 4E-binding motif. Yeast strain YGC034 (eap1::TRP1) was transformed with vector YEp352 alone or with the same vector expressing either HA-Eap1p or the HA-Eap1p[Y109A] mutant. Yeast were grown to exponential phase and lysed by the glass bead method. Protein (100 μg) was incubated with anti-HA monoclonal antibody before the addition of protein G-Sepharose beads. Following extensive washings, the bound proteins were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted against anti-HA antibody (to detect HA-Eap1p; indicated by a dot) (A) or anti-yeast eIF4E monoclonal antibody 9B12 (B). Load, an equal amount of extract used for the coimmunoprecipitation loaded directly onto the gel; Free, proteins remaining in supernatant following immunoprecipitation; Bound, proteins adsorbed to the resin. IgG, immunoglobulin G heavy chain. Sizes of standards are indicated in kilodaltons.