Fig. 3. SNX9 exhibits phospholipid binding and powerful lipid tubulating activity in vitro. (A) Nitrocellulose filters spotted with various phospholipids (100 pmol of each phospholipid as indicated in this figure) were incubated with purified 0.5 µg/ml of GST-SNX9 and the binding of each protein to the specific phospholipid was detected with an anti-SNX9 antibody. (B) Synthetic PE and PC liposomes supplemented with 10% of the indicated lipid were incubated with SNX9, sedimented and stained with Coomassie Blue. Three independent experiments were performed and the protein intensity was measured. S, supernatant; P, pellet. (C) Negative stain electron microscopy of liposome composed of a brain lipid extract after addition of recombinant SNX9. Scale bar: 300 nm. (D) Lipid droplets are rehydrated to form a lipid bilayer sheet with solution containing either GST-alone or GST-tagged SNX9 during microscopic observation. Time-lapse images of GST-control (top) and GST-tagged SNX9 (bottom) using differential interference contrast (DIC) microscopy. Dotted lines indicate the edges of membrane sheet. Scale bars: 40 µm.