Fig. 2. Effect of expression of SNAP-25 constructs on exocytosis in BoNT/E treated PC12 cells. The cells were co-transfected with a plasmid encoding growth hormone (GH) along with control vector (pcDNA3), plasmid encoding toxin-resistant mutant (Em2) or Em2 containing additional mutations as indicated. (A) Expression of SNAP-25 constructs demonstrated by blotting with anti-HA three days after transfection. (B) Transfected cells were permeabilised with 20 µM digitonin for 6 minutes and treated with recombinant BoNT/E light chain for 40 minutes. The cells were then challenged with 0 or 10 µM Ca²⁺ as indicated and GH release over a 20 minute period assayed. Total GH content for each well was determined and GH release is shown as a percentage of total GH expressed.