Fig. 2.

CpxI stimulates SNAREpin formation/assembly. (A) v-SNARE liposomes were modified with membrane-impermeable amino-reactive succinimidylester-activated biotin to label the pool of surface-exposed VAMP2 molecules selectively. These liposomes were incubated with t-SNARE liposomes in the absence or the presence of CpxI to allow SNAREpin formation for distinct periods of time at 4 °C. Subsequently, an excess of tetanus toxin was added, and the incubation continued for an additional 2 h at 4 °C to remove VAMP2 molecules not incorporated into SNAREpins. The reaction was stopped by the addition of SDS-containing sample buffer. (B) The toxin-resistant biotinylated VAMP2 was quantitated by Western blot analysis using antibiotin antibodies (details in Material and Methods). Error bars = SEM (n = 2).