Fig. 3. Cdc42p functions up to the docking stage. (A) Antibody inhibition. The indicated amounts of affinity-purified antibodies to Cdc42p were added to standard fusion reactions without cytosol. After pre-incubation for 10 min on ice, ATP was added and fusion was measured after 70 min at 27°C. (B) Kinetic analysis. Standard fusion reactions without cytosol were started. After the indicated times at 27°C inhibitors or control buffer were added. The samples were left on ice for 10 min. Then they were transferred to 27°C or left on ice for the remainder of the 70 min reaction period. Finally, fusion activity was assayed. Inhibitors used were: anti-Sec18p, 2 µM; Gdi1p, 5 µM; anti-Vam3p, 2 µM; Mastoparan, 20 µM; anti-Cdc42p, 8 µM.