Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

Rathore, Shailendra S.; Liu, Yinghui; Yu, Haijia; Wan, Chun; Lee, MyeongSeon; Yin, Qian; Stowell, Michael H.B.; Shen, Jingshi

GST pull-down assay-Full-length ternary SNARE complexes were assembled as previously described (Shen et al., 2007) . GST-Munc18-1 was expressed in E. coli using the pGEX4T-3-Munc18-1 plasmid and cell lysates were prepared using a protein binding buffer (25 mM HEPES [pH 7.4], 150 mM KCl, 10% glycerol, 1% CHAPS, and 1 mM DTT). SNARE complexes were added to GST-Munc18-1-expressing E. coli ly-sates. After incubation at 4°C for one hour, glutathione Sepharose beads were added to the lysates to bind GST-Munc18-1 and associated proteins. After washing three times with the proteinbinding buffer, protein complexes bound to the beads were resolved on SDS-PAGE and detected by immunoblotting using primary antibodies and horseradish peroxidaseconjugated secondary antibodies. The antibodies used in this work were polyclonal anti-Munc18-1 antibodies, monoclonal anti-syntaxin-1 antibodies, monoclonal anti-SNAP-25 antibodies, and monoclonal anti-VAMP2 antibodies.