Fig. 5. A peptide based on the eIF4E-binding sequence of sea urchin eIF4G inhibits formation of the SgIF4G-eIF4E complex and affects the first mitotic division of sea urchin embryos. (A) Incubation of a peptide based on the eIF4E-binding site of sea urchin eIF4G with recombinant eIF4E and SgIF4G fusion proteins inhibits their interaction. The fusion proteins, untreated (lane 1) or incubated with the SgIF4G peptide corresponding to the binding site on eIF4E (wild type) used at 50 µM (lane 2) or 20 µM (lane 3) or a scrambled peptide used at 50 µM (lane 4) or 20 µM (lane 5) were affinity-purified using an m⁷GTP column and were analysed by immunoblotting as described in Materials and Methods using an anti-GST antibody. Affinity-purified proteins were compared with the GST-fusion proteins loaded separately (lanes 6 and 7). The positions of the respective GST-fusion proteins are indicated by arrows on the right side of the panel. (B) A peptide based on the eIF4E-binding of sea urchin eIF4G affects the first mitotic division of sea urchin embryos. SgIF4G peptide corresponding to the binding site on eIF4E (wild type) or a scrambled peptide was introduced by microinjection at a final intracellular concentration of 20 µM into unfertilized eggs. The control corresponds to non-injected eggs. Cleavage was assessed under a light microscope at 150 minutes after fertilization. An average of 100 unfertilized eggs were injected for each compound in each experiment and error bars represent the standard deviation (± s.d.) of three independent experiments. Significance was assessed using Fisher's F-test and Student's t-test. ^*P<0.005, significant difference between eggs microinjected with the SgIF4G wild-type peptide, microinjected with the scrambled peptide and non-injected eggs.