Fig. 1: Membrane association of VAMP2 extravesicular domain in mammalian cells by in-cell NMR spectroscopy.

a Domain organization of VAMP2. P-rich NT: proline-rich N-terminal domain. JMD: juxta-membrane domain. TMD: transmembrane domain. R56 is conserved in the VAMP family which forms the zero ionic layer in the SNARE complex. b Scheme of in-cell NMR sample preparation. NMR-visible ¹⁵N-VAMP2(1–96) was delivered into mammalian cells by electroporation. Un-delivered proteins were washed off before NMR signal acquiring. c Estimation of VAMP2 quantity in cells by immunoblotting. Cells prepared for NMR were diluted 10 times before loading on gels. Known concentrations of VAMP2(1–96) proteins were loaded as standards. d Sub-cellular localization of VAMP2(1–96) by fractionation and immunoblotting. Fractions of the total lysate (Lys), cytosol (Cyto) and membrane (Mem) were validated by immunoblotting with antibodies of GAPDH, IRE1α, ACSL4, and VDAC to indicate cytosol, endoplasmic reticulum membrane, plasma membrane-associated membrane, and mitochondrial membrane, respectively. e Sub-cellular localization of VAMP2(1–96) by immunofluorescence staining. F-actin filaments beneath cell membranes were stained by FITC-phalloidin. Nuclei were stained by DAPI. f Overlay of 2D ¹H-¹⁵N NMR spectra of VAMP2(1–96) in solution (black), SH-SY5Y cells (blue) and HEK-293T cells (red). Disappeared crosspeaks in cells were denoted. *The peak was not able to be assigned. g Residue-resolved relative NMR intensity ratios (I/I₀) of VAMP2 (1–96) in cells to that in solution. Domain organization of VAMP2 extravesicular domain is indicated. h Residue-resolved ratios of ¹⁵N transverse (R₂, s⁻¹) to longitudinal (R₁, s⁻¹) relaxation rates of VAMP2(1–96) in HEK-293T cells (red) and in solution (black). Source data are provided as a Source Data file.