Fig. 2

Analysis of CXCR4 and US28 total and surface expression in mono-and coexpressing cells. a, b Radioligand-displacement studies to detect changes in CXCR4 and US28 surface expression were performed in transiently transfected COS-7 cells. Dose response curves represent the means ± SEM of seven independent experiments, each performed in duplicate. c-f For the ELISA - based analysis N-terminally FLAG-tagged CXCR4 and N-terminally HA-tagged US28, US28mutants and NTS1 were expressed in HEK293T cells. c Surface expression of CXCR4 was calculated as the signal ratio between permeabilized and non-permeabilized cells (reflected by FLAG-immunoreactivity) and normalized on the surface expression in CXCR4-only expressing cells. d The total expression of CXCR4 in mono- and coexpressing cells was calculated as a factor of FLAG-immunoreactivity in mock-transfected cells. e Surface expression of US28, US28 mutants and NTS1 in presence and absence of CXCR4 was calculated as the signal ratio between permeabilized and non-permeabilized cells (reflected by HA-immunoreactivity). f The total expression of US28, US28 mutants and NTS1 in presence and absence of CXCR4 was calculated as a factor of HA-immunoreactivity detected in mock-transfected cells. Columns represent means ± SEM from at least three independent experiments (n = 3–5), each performed in triplicates. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc test. (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant)