Fig. 2. SNAP-25 is required for Ca²⁺-dependent secretion in Neuro2A cells. (A) Homogenates of Neuro2A cells were analyzed by western blot with antibodies against Syntaxin-1, SNAP-25, VAMP-2 and Syndet. (B) Confocal immunofluorescence image of wild-type Neuro2A cells stained with antibodies against SNAP-25. (C) Confocal immunofluorescence image of Neuro2A cells expressing myc-tagged SNAP-25A stained with antibodies against myc. Bar, 15 µm. (D) Neuro2A cells were transiently transfected with POMC-ß-Gal and pcDNA3.1 or with POMC-ß-Gal and BoNT/E-pcDNA3.1. Ca²⁺-dependent ß-Gal release is measured as described in Fig. 1G. The average is calculated from the data of four independent experiments done with triplicate samples. (E) Neuro2A cells were transiently transfected with pcDNA3.1 (-) or BoNT/E-pcDNA3.1 (+) alone (lanes 1-4) or in combination with Syndet-pcB7 (lane 5 and 6) and Syndet-Delta-BoNT/E-pcB7 (Koticha et al., 1999) (lane 7). Syndet-Delta-BoNT/E protein expressed in Neuro2A cells is a truncated protein that is identical to the expected product of Syndet digested by BoNT/E (arrowhead) (Washbourne et al., 2001). The western blot was probed with anti-SNAP-25 and anti-Syndet antibodies, as indicated.

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