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Fig. 1. 4E-BP peptide affects eIF4E–4E-BP association and the first mitotic division of sea urchin embryos. (A) Treatment of extracts obtained from unfertilized eggs with the 4E-BP peptide leads to dissociation of eIF4E from 4E-BP. Control extract (lane 1) or extract treated with 10 µM variant eIF4E-binding peptide (lane 2) or 10 µM eIF4E-binding peptide (wild type) (lane 3) were incubated for 1 hour at 4°C. After incubation, eIF4E was purified from each extract using 50 µl m⁷GTP columns as described in the Materials and Methods. Proteins bound to the beads were separated by SDS-PAGE and subjected to western blotting using antibodies to eIF4E (top panels) or 4E-BP (bottom panels). Data are representative of three independent experiments. (B) Microinjection of 4E-BP peptide into unfertilized eggs impedes the first mitotic division triggered by fertilization. The eIF4E-binding peptide (wild type; white bar) or variant eIF4E-binding peptide (light grey bar) were injected at a final intracellular concentration of 10 µM. Control corresponds to unfertilized eggs microinjected with buffer (dark grey bar). Cleavage was scored by observation under a light microscope at 180 minutes after fertilization. An average of 100 unfertilized eggs were injected for each compound in each experiment and vertical bars represent the standard deviation of three independent experiments. Significance was assessed using Fisher's F-test and Student's t-test. An asterisk indicates a significant difference between eggs microinjected with eIF4E-binding peptide (wild type) and eggs microinjected with variant eIF4E-binding peptide or control (* corresponds to P<0.01).

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