Nbd pa

Phospholipids: POPC (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine, 850457C), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine, 850757C), POPA, E. coli cardiolipin (841199), 18:1-12:0 NBD-PA (1-oleoyl-2-(12-[(7-nitro-2-1,3- benzoxadiazol-4-yl)amino]dodecanoyl)-sn-glycero-3-phosphate, 810176C), 18:1-12:0 NBD-PS (1-oleoyl-2-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl) -sn-glycero-3-phosphoserine, 810195C), and Egg Liss-Rhod-PE (L-α- phosphatidylethanolamine-N-(lissamine rhodamine B sulfonyl), 810146C) were obtained from Avanti Polar Lipids. Lipids in stock solutions in chloroform were mixed at the desired molar ratio, and the solvent was evaporated. The lipid film was hydrated in appropriate buffer. The lipid suspension was incubated at room temperature for 30 min and extruded 20 times through polycarbonate 0.1-μm filter using a mini-extruder (Avanti Polar Lipids).

Lipids dissolved in chloroform were mixed at the desired molar ratio, and the solvent was evaporated by nitrogen gas. The lipid was hydrated with buffer B (20 mM HEPES pH 7.0 and 150 mM NaCl). After five freeze and thaw cycles with liquid nitrogen, liposomes were extruded through a mini-extruder (Avanti Polar Lipids) with 100 nm polycarbonate filter^{56 (link)}.
All lipids used in this study were purchased from Avanti Polar Lipids: PS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine), PC (1,2-dioleoylsn-glycero-3-phosphocholine), PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), PA (1,2-dioleoyl-sn-glycero-3-phosphate), PI3P (1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′-phosphate)), PI4P ((1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′-phosphate)), Cholesterol, NBD-PE, NBD-PS (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphoserine), NBD-PC, NBD-PA (1-palmitoyl-2-{12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl}-sn-glycero-3-phosphate), NBD-Ceramide (N-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-D-erythro-sphingosine), NBD-Cholesterol (5-cholesten-3ß-ol 6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproate), Rhod-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)), and DGS-NTA(Ni) (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]).

The procedure for the pulse-chase analysis of the S2 cells was modified from Miyata [27 (link)] and Tamura [54 (link)]. The S2 cells were incubated with NBD-PA, NBD-PC or NBD-PE (Avanti Polar Lipids, Inc.) for 20 min. The S2 cells were washed, then incubated for different periods of time. After harvesting, the total lipids in the cells were extracted and resuspended in chloroform/methanol (1:2, vol/vol). The lipid samples were separated by TLC on silica gel 60 F₂₅₄ plates (Merck,1.05729.0001) using a solvent system of chloroform/methanol/25% ammonia, 65:35:5 [26 (link)]. The TLC plates were detected with a Typhoon 9500 imager and the images were analyzed with Image J (1.51j8).