Figure 4

Regulation of eIF4E phosphorylation and eIF4F formation. A. Jurkat T cells were activated for 30 or 60 min by both αCD3 and αCD28. The primary T lymphocytes were activated for the indicated times with both αCD3 and αCD28 or with PMA. eIF4E was purified using m⁷GTP Sepharose, analyzed on a one-dimensional iso-electric focusing gel and detected by Western blotting. 4E and 4E-P indicate unphosphorylated and phosphorylated eIF4E respectively. B. 100 μg of total cell lysate from primary T cells treated for 1 h with αCD3 and αCD28 or PMA was analyzed by SDS-PAGE and Western blotting to detect 4E-BP1. (-) indicates untreated cells. The lane with 4E-BP1 from HeLa cell extract was obtained from a shorter exposure from the same blot. C. Formation of eIF4F was analyzed after 60 min activation of primary T cells with either αCD3, αCD28 or both. eIF4E was purified as described above and its association with eIF4G was analyzed by SDS-PAGE and Western blotting. An eIF4E blot was used to verify equal loading of all lanes. Similar results for eIF4E, eIF4G and 4E-BP1 were obtained in three independent experiments.