Fig. 1: Optimization of liposome-based transport assay for rGLUT5.

a Comparison of lipid extract effect on liposome reconstituted rGLUT5 ¹⁴C-d-fructose uptake, at 2 min. rGLUT5 specific uptake and non-specific uptake into empty liposomes are shown as bars, colored in black and white. Errors indicated per bar represent s.e.m. of three independent experiments. b Reconstitution efficiency of rGLUT5 into liposomes prepared using different lipid extracts: Brain 7, Brain 1, E. coli, Liver, Soya total and Soya PC (see “Methods”). The y-axis indicates the relative band densitometry of rGLUT5, and each black bar represents a relative densitometry band of rGLUT5 for each lipid extract used for reconstitution as shown in Supplementary Fig. 1e; error bars show the range of two independent reconstitutions. c Melting curve of rGLUT5 using lipid extracts as shown in Fig. 1a. As a control, the melting temperature of purified rGLUT5 with DDM added, but no lipid is shown in black. The data are normalized fluorescence mean ± s.e.m. of n = 3 independent experiments. d The melting temperatures (T_M) for rGLUT5 in the presence of different lipid extracts, as calculated from the curves in panel (c). Error bars indicate mean ± s.e.m. of n = 3 independent experiments. e Time course uptake of ¹⁴C-d-fructose by rGLUT5 in proteoliposomes and empty liposomes made using brain-fraction-seven represented as empty circles and squares, respectively. Errors bars represent s.e.m. of three independent experiments. f Zero trans kinetics of rGLUT5 d-fructose transport using brain-fraction-seven proteoliposomes. All data points at their respective d-fructose concentration were measured after 50 s, and data were fitted using a non-linear Michaelis–Menten function by GraphPad prism. Error bars indicate mean ± s.e.m. of n = 3 independent experiments.