Fig. 7. An eIF4G–PABP complex displaces maskin from eIF4E. (A) A peptide derived from eIF4G that is necessary for the binding of this protein to PABP was added to an egg extract, as was a control peptide of irrelevant sequence. The extract was then applied to an affinity column containing (lanes 1 and 2) or lacking (lane 4) recombinant His-tagged PABP. Following extensive washing, the columns were eluted with SDS and probed on a western blot for eIF4G and PABP. Lane 3 shows the profile of eIF4G in the extract prior to chromatography. (B) Various concentrations of the eIF4G-derived peptide and the control peptide were injected into oocytes that were subsequently incubated with progesterone. The incidence of oocyte maturation was then scored by the appearance of a white spot at the animal pole. (C) Oocytes injected with various concentrations of the control peptide or the eIF4G-derived peptide were incubated with progesterone, scored for the appearance of the white spot and then analyzed for cyclin B1 accumulation by western blotting. The control peptide-injected oocytes had matured whereas the eIF4G-derived peptide-injected oocytes had not. The asterisk denotes a non-specific band that serves as a loading control. (D) Oocytes injected with the control and eIF4G-derived peptides were incubated with progesterone, homogenized, mixed with free GTP and applied to a cap column. Following extensive washing, the column was eluted with SDS and probed on western blots for maskin and eIF4E. Maskin in the load fraction was also analyzed on a western blot. The control peptide-injected oocytes had matured whereas the eIF4G-derived peptide-injected oocytes had not. (E) Oocytes injected with poly(A) or the eIF4G peptide were also injected with maskin antibody and subsequently used for a cyclin B1 western blot. The asterisk denotes a non-specific band.