Figure 1.

(A) Competition test of a mixture of eIF4E [∆1–46] and eIF4E[∆1–46/∆216–230] for m7GTP cap analog (A) and VPg (B) binding. (A) A mixture of both species was loaded on an m7GTP affinity column. The fraction eluted in 0.5 M KCl was analyzed by SDS-Page and Western blot. (B) A mixture of both species was incubated with (His)₆-VPg and a Ni²⁺ IMAC was performed. The eIF4E [∆1–46/∆216–230] species was not retained (flow through). The eIF4E [∆1–46] species co-eluted with (His)6-VPg. Elution (250 mM imidazole). Fractions were analyzed by SDS-Page (Co) and Western blot Wt). Western blot revelation: lettuce eIF4E polyclonal antibodies. Subsequent analysis confirmed the identity of the two eIF4E species. The bands were cut from the gel and analyzed by mass spectrometry. Alternatively, they were transferred on a PVDF membrane and submitted to N-terminal sequencing. Both forms displayed the same N-terminal amino acid sequence.