Fig. 4. The 40 kDa protein associates with eIF4E. (A) Total extract obtained from unfertilized eggs was subjected either to western blotting using 4E-BP2 antibody (lane 1) or to far-western analysis (see Materials and Methods) using ³²P-labelled mouse recombinant HMK-eIF4E as a probe (lane 2). (B) Western blot and far-western analyses of the purified 40 kDa protein. Proteins from unfertilized egg extracts were immunopurified using 4E-BP2 antibodies coupled to Sepharose beads (1) or using a m⁷GTP column (3). The proteins fixed on the beads were resolved either by western blotting using 4E-BP2 antibodies (left panel) or by far-western analysis using an eIF4E-HMK probe (right panel). 4E-BP2-coupled Sepharose beads alone (lane 2) or m⁷GTP-column beads alone (lane 4) were used as controls. Arrows point to the positions of 4E-BP and the 40 kDa protein. Data are representative of at least three independent experiments.