Supplementary Figure 4: Correlation plots of syt linker mutant t-SNARE or PS binding activity versus evoked transmission.
From: Linker mutations reveal the complexity of synaptotagmin 1 action during synaptic transmission
(a) t-SNARE binding activity (defined as the mole of syt bound per mole of syntaxin (syx)) of WT or linker mutant cytoplasmic domains (C2AB) was measured using a co-flotation assay. The final [Ca2+]free was 1 mM; [EGTA] was 0.2 mM. Four independent experiments were carried out. (b) t-SNARE binding activity of WT or linker mutant forms of C2AB were plotted versus eEPSC amplitude from Fig. 2b. (c) Binding of WT or linker mutant forms of C2AB to PS-bearing liposomes was monitored using a co-sedimentation assay; depletion of the supernatant was monitored via SDS-PAGE and staining with Coomassie blue (the staining intensity reflects free C2AB in the supernatant), and these data were used to calculate the amount of bound material, in the absence (–; 0.2 mM EGTA) or presence (+) of 1 mM [Ca2+]free. Three independent experiments were carried out. (d) PS binding activity (defined as the apparent Kd for lipid; note that [lipid] and not [liposome] values were used, thus resulting in apparent Kd values in the mM range) of WT or linker mutants were plotted versus the eEPSC values that were, again, from Fig. 2b; error bars represent SEM. Equations and p-values for the linear regressions are listed in Supplementary Table 1. For clarity, the upper and lower portions of each gel (i.e. regions lacking proteins), were cropped.
