Figure 5.

Ca²⁺-dependent inhibition of the binding of native synaptotagmin with syntaxin by the synprint peptide. Coimmunoprecipitation of syntaxin and synaptotagmin was measured as described for Fig. 4 in the presence of 0.5 μM of His-L_II–III(718–963) from α_1B or 0.5 μM of His-L_II–III(670–800) from the L-type α_1S under Ca²⁺-buffering conditions containing 0, 10, 20, and 1000 μM of free Ca²⁺ as indicated. The immunoprecipitates were analyzed by SDS/PAGE, immunoblotted with anti-synaptotagmin (1D12) and anti-syntaxin (10H5) antibodies, and visualized with rabbit anti-mouse IgG coupled to horseradish peroxidase and enhanced chemiluminescence detection. The amount of immunoprecipitated synaptotagmin was measured by densitometry and compared with control samples in the absence of competing peptides. The migration positions of synaptotagmin and mouse IgG in the immunoprecipitation are indicated.