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. 2002 Sep 2;158(5):929–940. doi: 10.1083/jcb.200112081

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Copyright © 2002, The Rockefeller University Press

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Figure 6. — v_N-pep and v_C-pep act directly upon the coiled-coil region of the t-SNARE. The NRD of syntaxin was removed from t-liposomes (32 μg protein before digestion) by proteolytic digestion at a thrombin site introduced at residue 181 of syntaxin (Parlati et al., 1999). The resulting-t-SNARE liposomes (TΔNRD) were incubated with v_C-pep (A) or v_N-pep (B) for 4 min before initiating the fusion assay by addition of v-liposomes (V). The control uncleaved t-liposomes (T) were incubated with preinactivated thrombin. The symbols in all panels are as follows: T + V (○), T + V + peptide (•), TΔNRD + V (□), TΔNRD + V + peptide (▪), TΔNRD + V + v_C-pep + v_N-pep (▴). (C) Protein pattern of t-liposomes analyzed by SDS-PAGE and Coomassie blue staining. Thrombin cleavage generates the NRD and H3 regions of syntaxin. SNAP-25 and syntaxin in the lumen of the liposomes is protected from thrombin.