Fig. 4. Sec17p/α-SNAP release is not compromised by anti-Cdc42p. Standard fusion reactions (3-fold volume) without cytosol were incubated for 70 min without ATP on ice (0 min) or with ATP at 27°C for 15 or 70 min. Where indicated, 6 µM anti-Cdc42p was added. Reactions were chilled on ice and centrifuged (10 000 g, 10 min, 4°C). The supernatants (S) were recovered and the pellets (P) resuspended in 90 µl of PS buffer. All samples were trichloroacetic acid precipitated and processed for non-reducing SDS–PAGE and western blotting with the indicated antibodies. Alkaline phosphatase (Pho8p) serves as a membrane integral vacuolar marker.