FIGURE 6.

SNAP25B reduces the extension of Sx1A-Sb2 interactions. (A) Sx1A functionalized tips (Sx1A-H6) were preincubated with GST-SNAP25B. As revealed by indirect immunochemistry (middle, bright-field images; bottom, fluorescence images) only tips preincubated with GST-SNAP25B (+) show positive immunoreactivity. (B) The retraction part of typical force-distance curves and distribution of measured extensions (C) when tips were preincubated either with GST-SNAP25B (top), GST (middle), or where tips were functionalized with truncated Sx1A_178-266-H6, incubated with GST-SNAP25B, and then used to probe Sb2 functionalized coverslips (bottom). (D) Summary of all experiments shown in Figs. 1–6 indicate that there is no significant difference in the rupture force in any condition tested (top), whereas the extension measurements (bottom) are an invaluable tool in the assessment of the functional role of individual SNARE proteins. Arrowheads in C indicate mean values. Bars in D represent mean ± SE of 138–456 events. Solid bars show data acquired on Sb2-H6 functionalized coverslips probed with Sx1A-H6 functionalized tips, whereas the hatched bars indicate coverslips tested with the truncated form of Sx. Statistical significance was established by a one-way ANOVA followed by a post-hoc Scheffeé's comparison at P < 0.05 (*) or P < 0.01 (**). Scale bar, 30 μm. Retraction velocity, 1.6 μm/s. Drawing in A is not to scale.