Fig. 5. The new 40 kDa eIF4E-binding protein presents behaviour similar to that of the sea urchin 4E-BP already identified. (A) Rapamycin inhibits the disappearance of the 40 kDa protein following fertilization. The total amount of the 40 kDa protein from unfertilized eggs (lane 1) and from untreated (lane 2) or rapamycin-treated (lane 3) eggs obtained 60 minutes following fertilization was analysed by western blotting using 4E-BP2 antibodies (top panel). A western blot anti-actin was also performed (bottom panel) as loading control. (B) Rapamycin inhibits the dissociation of the 40 kDa protein from eIF4E. Proteins affinity-purified using m⁷GTP columns prior to fertilization or at the indicated times following fertilization of untreated (left) or rapamycin-treated (right) eggs were subjected to western blotting using anti-4E-BP2 (top) or anti-eIF4E (bottom) antibodies. The data are representative of at least five independent experiments. (C) Treatment of extracts obtained from unfertilized eggs with the 4E-BP peptide also leads to the dissociation between eIF4E and the 40 kDa protein. Control extract (lane 1) or extract treated with 10 µM variant eIF4E-binding peptide (lane 2) or 10 µM eIF4E-binding peptide (lane 3) were incubated for 1 hour at 4°C. After incubation, eIF4E was purified using a m⁷GTP column. Proteins bound to the beads were separated by SDS-PAGE and subjected to western blotting using 4E-BP (top panels) or eIF4E (bottom panels) antibodies.