Fig. 2. Sea urchin eIF4G is modified following fertilization and interacts with eIF4E in vivo. (A) Specificity of SgIF4G antibodies. Supernatants obtained from low-level speed centrifugation of egg homogenates were prepared prior to (lanes 1, 3, 5 and 7) or following (lanes 2, 4, 6 and 8) fertilization. Identical amounts of extract proteins (10 µg) were resolved by 7.5% SDS-PAGE and subjected to western blot analyses using anti-SgIF4G antibody (lanes 1 and 2), corresponding pre-immune serum (lanes 3 and 4), anti-SgIF4G antibody pre-incubated with 1 µg recombinant GST-SgIF4G (lanes 5 and 6) or anti-SgIF4G antibody pre-incubated with 1 µg of GST (lanes 7 and 8); anti-actin antibody was used as loading control. (B) The multiple isoforms of SgIF4G associate with the GST-tagged mIF4E. Proteins extracted from unfertilized eggs or eggs 60 minutes post fertilization were incubated with GST alone or GST-tagged mIF4E proteins bound to glutathione-Sepharose beads as described in Materials and Methods. Precipitates were analysed by western blotting using anti-SgIF4G (top panels) and anti-GST (bottom panels) antibodies. Input represents 20% of total protein used for pull-down. (C) SgIF4G co-purifies with sea urchin eIF4E. Total-extract proteins (left panels) or affinity-purified proteins using an m⁷GTP column (right panels) prior to or 60 minutes post-fertilization were subjected to western blot analyses using anti-SgIF4G (top panels), anti-actin (middle panels) or anti-eIF4E (bottom) antibodies. (D) Sea urchin eIF4E coimmunoprecipitates with SgIF4G after fertilization. Proteins extracted from unfertilized eggs or eggs 60 minutes post fertilization were immunoprecipitated with SgIF4G, resolved by SDS-PAGE and analysed by western blotting using anti-rabbit eIF4E (lower panel) and anti-SgIF4G antibodies (upper panels). The arrow indicates IgG recognized by secondary antibodies. Protein A Sepharose beads with anti-SgIF4G but without proteins and protein A Sepharose beads with proteins but without anti-SgIF4G were used as controls.