Fig. 3

Binding of soluble Syncytin-1 to FuTraP-ASCT2 variants expressed on the cell surface. a Schematic representation of the vector expressing the soluble form of Syncytin-1 (sS1). sS1 was cloned downstream of the second splice acceptor (SA) of replication-competent avian retrovirus RCASBP(B), which permits propagation in cell culture and shedding of sS1 into the supernatant. sS1 consists of the signal peptide of Env(A) (sigPE(A)) followed by the in-frame receptor-binding domain of Syncytin-1 (SU(S1)-RBD), Tobacco Etch Virus protease cleavage site (T), and rabbit heavy chain of IgG (rIgG). sigPE(A) is composed of six amino acids from the retroviral Gag coding sequence (blue) followed by 56 amino acids from Env(A) (light yellow). The numbering corresponds to the original protein sequences. Long dashed lines illustrate two possible spliced products using the same splice donor (SD) and two alternative SA. The sS1 protein is produced to the supernatant (together with RCASBP(B) virus) and used for the cell surface labelling of ASCT2 variants and for inhibition experiments (Figs. 4 and 5). b Labelling of living cells with sS1. DF-1/LgBiT cells expressing either variants of FuTraP were incubated with sS1, and its binding was visualised by staining with anti-rabbit IgG antibody conjugated to Alexa Fluor 594. Median of Alexa Fluor 594 fluorescence intensity was measured by flow cytometry for individual FuTraP variants and normalised to the wild-type FuTraP-hASCT2-wt (Y-axis). LgBiT cells expressing the S1 allele of chicken Tvb (ggTvb^S1) or human EAAT1 (hEAAT1) represent the negative control (NC). Results of three independent experiments performed in duplicates are shown. Data is shown as means ± standard errors, **p < 0.01