Fig. 7. The two helices of SNAP-25 can function independently of each other in regulated exocytosis. (A) A SNAP-25 construct called S25A/ER-82-206-pcB7, lacking the N-terminal helical region and containing amino acids 82-206 was generated. (B) Confocal immunofluorescence image of Neuro2A cells expressing myc-tagged S25A/ER-82-206 stained with antibodies against myc. Bar, 15 µm. (C) Ca²⁺-dependent secretion of ß-Gal activity (percentage of total activity in cells) was derived as in Fig. 5 A. ß-Gal secretion is measured in G14 cells stably expressing BoNT/E and transiently transfected with POMC-ß-Gal and pcB7 or S25A/ER-82-20-pcB7. Ca²⁺-dependent release of ß-Gal (as in Fig. 1G) is calculated from the data of four independent experiments done with triplicate samples as in Fig. 1. (D) SNARE complexes derived from G14 cells transiently transfected with POMC-ß-Gal together with either pcB7 or S25A/ER-82-206-pcB7 were detected as described in Fig. 6. The total amount of S25A/ER-82-206 expressed is shown in the lower panel probed with anti-myc antibodies.