Fig. S2.

eIF4E/m⁷GTP/4E-BP1 protein complex preparation and limited proteolysis. (A) SDS/PAGE analysis of recombinant eIF4E/4E-BP1 full-length protein complex expressed in bacterial cells and purified over an m⁷GTP-Sepharose column. (B) SDS/PAGE analysis of the purified fractions obtained after S75 gel filtration chromatography, containing eIF4E/m⁷GTP/4E-BP1 after TEV cleavage. The same procedure was used to express and purify other complexes mentioned in this study. (C) Protein eIF4E and 4E-BP1 full-length are indicated. Fragments generated (a, b, and c) from the eIF4E/4E-BP1 complex digested for different time by subtilisin were separated by HPLC and submitted to mass spectrometry analysis (LC-MS/MS).