Figure 7. Wild type Sg4E-BP and the S/T-E variant inhibit eIF4E/eIF4G association with the same efficiency.

Wild type (A) and the 4xE variant of Sg4E-BP (B) inhibit eIF4E/eIF4G association. The m⁷GTP sepharose beads was incubated (lanes 1–7) or not (lanes 8–9) with GST-mIF4E (eIF4E). Then, recombinant proteins GST-Sg4E-BP WT (WT, A) or GST-Sg4E-BP WT 4xE (4xE, B) and GST-SgIF4G (eIF4G) were added separately for lanes 1–2 and lanes 8–9, and together for other lanes (3-4-5-6-7). We used the same amount of GST-Sg4E-BP and GST-SgIF4G in lane 5, a 2-fold ratio in lanes 4 and 6 and a 10-fold ratio in lanes 3 and 7. Proteins were affinity-purified using m⁷GTP sepharose beads and were analysed by immunoblotting as described in Materials and Methods using an anti-GST antibody. Affinity-purified proteins were compared with the GST-fusion proteins loaded separately (lanes 10–12). Inputs represent 10% of the volume used in the experiment.