Fig. 6. Rap1 activation in response to hyperosmolarity. Ax-2 cells were withdrawn at the indicated times (lanes 0, 5, 10, 20, 30 and 60 minutes) after the addition of 0.4 M sorbitol and lysed. Cell lysate (100 µg) was incubated with GST-RalGDS(RBD)-glutathione-Sepharose beads, and the amounts of bound Rap1 were determined by western blotting. A sample was also taken immediately following the wash procedure, that is, immediately before the 1 hour adaptation in KK₂ prior to sorbitol addition (lane OK).