Skip to main content
. 2017 Apr 28;58(5):962–973. doi: 10.1194/jlr.M076133

Fig. 2.

Fig. 2.

Cell-free expression and functional analysis of human SMS family members. A: Schematic outline of the wheat germ-based dialysis mode for cell-free translation of SMS-V5 mRNA. Unless indicated otherwise, translation reactions were supplemented with liposomes containing equal amounts of phospholipid head group donors, PC and PE. B: Translation reactions with or without SMS-V5 mRNA were subjected to immunoblot analysis using anti-V5 antibody. Known amounts of a 75 kDa V5-tagged reference protein, pRef-V5, were included to allow quantification of cell-free-produced SMS-V5 protein. Note that translation reactions with SMS-V5 mRNA in each case yielded an immunoreactive protein of the expected size. C: SMS2-V5 mRNA was translated in the absence or presence of 2 mM liposomes. Translation reactions were subjected to density gradient fractionation and immunoblotting using anti-V5 antibody. D: SMS2-V5 mRNA was translated in wheat germ extract in the presence of the indicated amounts of liposomes. Cell-free produced SMS2-V5 was incubated with NBD-Cer and reaction products were analyzed by TLC. Expression of SMS2-V5 was verified by immunoblotting using anti-V5 antibody (bottom). E: TLC analysis of reaction products formed when SMS2-V5 produced in the presence of 2 mM liposomes was incubated with NBD-Cer for the indicated period of time. Sensitivity of detection was increased 10-fold compared with (D) to visualize SMS2-mediated production of NBD-CPE. Migration of an unidentified fluorescent lipid that was also present in reactions lacking SMS2 is marked by an asterisk. F: Quantification of reaction products formed by cell-free-produced SMS2-V5 when incubated with NBD-Cer for the indicated period of time. G: Functional analysis of cell-free-produced SMS1, SMS1CPE, and SMS2. TLC analysis of reaction products formed when the indicated SMS enzymes produced in the presence of 2 mM liposomes were incubated with NBD-Cer. SMS expression was verified by immunoblotting using anti-V5 antibody (bottom). Data shown are representative of three independent experiments.