Fig. 2.

Binding efficacy of rcIL-31 to membrane-anchored receptors, cIL-31RA and cOSMRβ. a–c Flow cytometric analysis of the binding ability of rcIL-31 to cIL-31RA, cOSMRβ and hOSMRβ, which were fused with EGFP at their C-terminals. Biotinylated rcIL-31 proteins (rcIL-31-Biotin), at a concentration of 20.0 μg/ml, were incubated with HEK293T cells, which were expressed with cIL-31RA-EGFP, cOSMRβ-EGFP or hIL-31RA-EGFP on the cell membrane. The binding signals were picked up by PE/Cyanine7-labeled streptavidin. The data were representative of three independent experiments with similar results. d Binding curves of rcIL-31 to membrane-expressed cOSMRβ and cIL-31RA were established based on the binding percentages at a series of dilutions of rcIL-31-Biotin. The binding percentage was calculated based on the ratio of rcIL-31-Biotin- and EGFP-fusion protein double-positive cells to all EGFP-fusion protein-positive cells. The concentrations corresponding to 50% of the maximal effect (EC₅₀) were calculated based on these curves built with GraphPad software. The experiment was repeated three times, and an error bar indicates the mean standard deviation (n = 6)