Sequence alignment NME family members. (A) Amino acid sequence alignment of the N-terminal region of human NME family members. The N17 amino acid region of NME3 is marked in yellow. These sequences were analyzed with EMBL-EBI Clustal Omega Multiple Sequence Alignment. (B) Sequence alignment of the N-terminal region of NME3 from different organisms. (C) Glutaraldehyde crosslinking experiment. To analyze the oligomerization status of recombinant NME3^WT-His and NME3^ΔN-His, 4 μM purified proteins were subjected to crosslinking using glutaraldehyde, forcing the formation of covalent bonds, and detected by Western blotting. (D) Liposome binding ability of N17-GFP. To examine the direct binding between N17 and lipid, N17-GFP was purified from N17-GFP transfected HeLa cells with GFP-trap, and then subjected to liposome binding composed of 1% rhodamine-PE, 79% PC, and 20% indicated lipid. After PBS wash, bound liposomes were quantified as remained fluorescent intensity. Upper panel shows the fluorescent intensity of bound liposomes, where the lower gel shows the amount of protein in each binding reaction. N = 3. (E) Size distribution of PC liposomes prepared by sonication (SUV) or extrusion through membrane with 50 nm pore. Data are expressed as mean ± SD of three independent experiments and analyzed with one-way ANOVA. **P < 0.01; ns, not significant. Source data are available for this figure: SourceData FS2.