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. 2022 Jan 10;13:97. doi: 10.1038/s41467-021-27658-x

Fig. 6. GPR182 binds and endocytoses chemokine CXCR3 ligands.

Fig. 6

a, b WT CHO cells and CHO cells stably transfected with human GPR182 (GPR182 + CHO) were stained for human CXCL9-FLAG binding at 4 degrees. Cells were pre-incubating with purified human CXCL9 to assess its blocking capacity (a). The addition of different concentrations of unlabeled CXCL9 was used to quantify the IC50 and Kd (b). n = 3 replicate samples. Representative data from at least three independent experiments. c WT 293T cells and 293T cells transiently transfected with mouse GPR182 were stained for mouse CXCL9-FLAG binding at 4 degree. n = 3. Representative data from two independent experiments. d GPR182 + CHO cells were stained for hCXCL9-AF488 at 4 degree. Cells were pre-incubated with different concentrations of CXCL10 or CXCL11 to assess their blocking capacities. n = 3 replicate samples. Representative data from two independent experiments. e, f WT and GPR182 + CHO cells were incubated with CXCL9-AF488 at 37 degrees for 20 min, followed with fixation and costaining with wheat-germ agglutinin (WGA). CXCL9-AF488 after incubation with WT or GPR182 + CHO cells was determined by confocal microscopy. n = 10 per group (e). The location of GPR182 with or without CXCL9 incubation was determined by confocal microscopy (f). Representative data from two independent experiments. g–j WT and GPR182 + 293T cells were incubated with CXCL9 for 20 min at 37 degrees followed with fixation and staining for GPR182 and Clathrin HC (g), Caveolin-1 (h), EEA1 (i), or LAMP1 (j) before confocal microscopy. Representative data from two independent experiments. P-value from two-sided paired t-test (e). Error bars represent SEM.