Fig. 5.

Analysis of binary and ternary SNARE protein interactions via ligand overlay blotting. A, Wild-type and mutant VAMP-2 proteins (100 ng each) were resolved by SDS-PAGE and transferred to nitrocellulose. After an incubation with 2 μg/ml of either syntaxin 1A (upper) or both syntaxin 1A and SNAP-25 (lower), the blots were probed for syntaxin 1A immunoreactivity, as described in Materials and Methods.B, Blots identical to those described inA were incubated with 2 μg/ml of either SNAP-25 (upper) or both SNAP-25 and syntaxin 1A (lower) and probed for SNAP-25 immunoreactivity.C, Relative binding intensity of syntaxin 1A (open bars) and SNAP-25 (solid bars) to immobilized VAMP-2 proteins, as determined by quantitation of the blots shown in the lower panels of A andB. All values were normalized to those for wild-type VAMP-2.