Membrane sheets are generated from PC12 cells expressing GFP-SNAP25 alone (control) or together with Stx-full/Stx-ΔS. Endogenous syntaxin/syntaxin constructs are recognized by an antibody raised against the molecule’s N-terminus, visualized by an AlexaFluor594-coupled secondary antibody; GFP-SNAP25 is visualized by an ATTO647-labelled GFP nanobody. (A) Representative images from the syntaxin 1A and SNAP25 channels of the control (only endogenous syntaxin), Stx-full (+ additional syntaxin), and Stx-ΔS (+ additional syntaxin with a shortened SNARE domain) condition. For magnified views see Figure 4—figure supplement 4. Top right corner, average Pearson correlation coefficient (PCC) between the two channels; for statistics see Figure 4—figure supplement 5. (B) Change of the syntaxin 1A staining pattern after Stx-full/Stx-ΔS expression. Staining intensity (region of interest [ROI] intensity), maxima size, and maxima density are related to the average control values (set to 100%). (C) SNAP25 staining pattern after Stx-full/Stx-ΔS expression. SNAP25 staining intensity (ROI intensity), clustering degree (relative standard deviation of the mean [rSDM]; 100% = 0.47), and maxima density are related to the control. For the relation between rSDM and Stx-full expression level, see Figure 4—figure supplement 6. Values are shown as box plots (n = 6–9 experiments, including three experimental preparations used as well for Figure 2; at least 15 membrane sheets were imaged per condition and experiment), showing the median, the 25th and 75th percentile (box), and the minimum and maximum value. Two-tailed paired Student’s t-test compare (1) control to Stx-full/Stx-ΔS and (2) Stx-full to Stx-ΔS; ns = not significant, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. After exchanging the spectral properties of the labels using ATTO647 for the syntaxin and ATTO594 for the SNAP25 channel, the same trends are observed. The syntaxin 1A maxima are sharper and more defined than SNAP25 maxima as well (Figure 4—figure supplement 7).