Fig. 1
From: Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor
pFuTraP vectors and their expression. a Scheme of the pFuTraP-hASCT2-wt vector. The vector was constructed using MAV LTRs, RCASBP encapsidation signal (Ψ), human ASCT2 fused with AcGFP, IRES, and iRFP713 fluorescent marker. b Scheme of the ASCT2 structure and alignment of mutants. Depicted are eight transmembrane helices and two helical hairpins (displayed in grey, numbered I-VIII and HP1-2, respectively), which divide the protein into intracellular (light green) and extracellular parts (orange). The longest extracellular loop (ECL2) is interrupted by short membrane regions (IVa and IVb) to the N-terminal (region N) and C-terminal (region C) parts. Region C is stabilised by two antiparallel beta-sheets and was proposed as a retroviral docking site. The amino acid sequences of ASCT2 mutants with deglycosylated and shortened region C used in this study are shown. The numbering corresponds to the protein sequence of wild-type ASCT2, the glycosylated asparagine N212 is shown in bold. c Scheme of the membrane topology of ASCT2 (modified from [26]). d iRFP713 and AcGFP expression from pFuTraP vectors. DF-1/LgBiT cells with ectopic expression of pFuTraP vectors were characterised by flow cytometry. The analysis was repeated three times in biological duplicates and representative dot plots of 3 × 104 cells are presented. The X-axis depicts the AcGFP fluorescence, Y-axis depicts the iRFP713 fluorescence. e iRFP713 fluorescence reflects the level of RNA produced from pFuTraP vectors. The MFI of iRFP713 of individual variants relative to the FuTraP-hASCT2-wt is plotted. f AcGFP fluorescence reflects the level of FuTraP protein produced from pFuTraP vectors. The MFI of AcGFP of individual variants relative to the FuTraP-hASCT2-wt is plotted. g Protein production from pFuTraP vectors. For each sample, the relative MFI of AcGFP (protein level) was normalised to the relative MFI of iRFP713 (mRNA level). NC, negative control (ggTvbS1 or hEAAT1). Data is shown as means ± standard errors, *p < 0.05, **p < 0.01, Mann–Whitney two-tailed test