Figure 8.

Analysis of the solubility of WT and mutant synaptotagmin-1 fragments in the presence of SNARE complex. Samples containing 10 µM C₂AB fragment (A,C) or 10 µM C₂B domain (B,D) were incubated with 10 µM (A,B) or 20 µM (C,D) SNARE complex for 5 min in the presence of 1 mM Ca²⁺. The soluble fractions (S) and the precipitates (P) were separated by centrifugation and analyzed by SDS PAGE followed by Coomassie blue staining. The positions of the C₂AB fragment (C₂AB), C₂B domain (C₂B) and SNARE complex (SC) are indicated. Note that the C₂AB fragment runs above the SNARE complex (panels A,C) but the C₂B domain runs below the SNARE complex (panels B,D) because of its smaller size. Note also that the KK and DN mutant C₂AB fragments run slightly differently than the WT C₂AB fragment, which most likely arises because the mutations change charged residues.