Fig. 5. GST-RalGDS(RBD) binds to the activated form of Rap1. (A) Ax-2 and pVEII-Rap1 (G12V) transformant cells were lysed, and the indicated amounts of cell lysate were incubated with 20 µg GST-RalGDS(RBD) that had been precoupled to glutathione-Sepharose beads for 1 hour at 4°C. The beads were pelleted by centrifugation, and the bound Rap1 was released by adding SDS sample buffer. The samples were fractionated by SDS-PAGE, and Rap1 was detected by western blotting using the Dictyostelium Rap1-specific antibody. (B) Bacterially expressed Rap1 was pre-equilibrated with 1 mM GDP (lanes 1, 2, 5 and 6) or 1 mM GTP (lanes 3, 4, 7 and 8) and incubated with GST-RalGDS(RBD) bound to glutathione-Sepharose beads (lanes 1-4) or GST bound to glutathione-Sepharose beads (lanes 5-8). The Sepharose beads were pelleted by centrifugation, and both the bound Rap1 in the pellets (lanes 2, 4, 6 and 8) and the unbound Rap1 in the supernatants (lanes 1, 3, 5 and 7) were fractionated by SDS-PAGE and detected by western blotting.