Fig. 1. Ca²⁺ induces the formation of multiple MgATP-dissociatable complexes in situ. Chromaffin cells were permeabilised with 20 µM digitonin in KGEP and maintained in the absence or presence of 20 µM Ca²⁺, with or without the inclusion of 2 mM MgATP. After 15 minutes, aliquots were removed and assayed for catecholamines. Cells in some wells were solubilised in Triton X-100 and used to estimate the total amount of catecholamine remaining and the amounts released (A) were expressed (±s.d. n=4) as a percentage of the total cell content, calculated by summing the amounts released with the level remaining. The remaining cells were lysed in KGEP, 2 mM PMSF was added and membrane-enriched fractions were isolated, as detailed in the Materials and Methods. The latter were dissolved in 50 mM Tris-HCl, pH 5.8 containing 1% SDS before adding 4x sample buffer and being subjected to SDS-PAGE. The separated proteins were either transferred directly to PVDF (B) or extracted from the gel by boiling in fresh sample buffer and re-electrophoresed before transfer (C). The PVDF membranes were blotted with antibodies raised against recombinant SNAP-25. Immunodetection of primary antibody binding was performed as described in Materials and Methods. Note that the left and right panels show signals for two different bands (M_r=25 K and 83 K, respectively).