(A) Domain diagram of the 20S complex consisting of NSF (orange, light blue, pink), αSNAP (yellow), and the SNARE complex composed of the cytoplasmic domain of syntaxin-1A (red, amino acid range 180–262), two SNARE domains of SNAP-25A (green, amino acid range 1–85 and 120–206) or full-length SNAP-25A (green, amino acid range 1–206), and the cytoplasmic domain of synaptobrevin-2 (blue, amino acid range 1–96) (Materials and methods). (B) SDS-PAGE gel of the 20S complex formed in the absence and presence of the native linker of SNAP-25A that connects its two SNARE domains. (C) The intensities of the protein bands in the SDS-PAGE gel were quantified using ImageJ (Schneider et al., 2012). Shown are mean values ± SD for protein band intensities of αSNAP divided by the protein band intensities of NSF from two independent gels prepared by the same samples. The protein band intensities from the boiled samples were used since the molecular weight of the SNARE complex without the S25 linker was only slightly higher than that of αSNAP, interfering with densitometry. Additionally, all protein intensities were background-subtracted using the intensity of a nearby selected rectangular area.