Fig. 3.

Distribution of (A) NSF and (B) β-SNAP in P2-GCV. A, NSF was still bound to the GCVs after the Mg²⁺-ATP treatment (5 mm MgCl₂/0.5 mm ATP on ice for 5 min; see Hong et al., 1994) (lanes 1 and2), similar to the case in SVs (lanes 3and 4) but not the Golgi apparatus (lanes5 and 6). After treatment, the sample was centrifuged at 41,000 rpm for 30 min. Lanes1, 3, and 5, pellet; lanes2, 4, and 6, supernatant. The supernatant and the pellet were subjected to SDS-PAGE and analyzed by immunoblotting using anti-NSF antibody. B, β-SNAP was not released from the membranes in GCV (lanes 1 and2) or in SV (lanes 3 and4), even in the presence of Mg²⁺-ATP (see above). Under the same conditions, however, β-SNAP was released from membrane in the Golgi apparatus (lanes 5 and6). Lanes 1, 3, and5, pellet; lanes 2, 4, and6, supernatant. The supernatant and the pellet were analyzed by immunoblotting using anti-β-SNAP antibody. Each protein amount was measured by densitometric assay of the immunostained bands.