Figure 2. (A) CXCR7 internalization depends on CCPs and is G protein-independent.

HEK293T cells were transfected with wt CXCR7 (and β-arrestin (319–418) were indicated) and cell surface levels of the receptor after CXCL12 stimulation was detected by ELISA using the CXCR7-specific antibody 11G8. Incubation with 0.4 M Sucrose was done 30 min prior and during stimulation. PTX was incubated overnight at 25 ng/ml final concentration. (B) β-arrestin1/2 knock-down prevents CXCR7 internalization. HEK293/CXCR7 cells transfected with control siRNAs (white bars) or pools targeting β-arrestin1/2 (filled bars), were stimulated with CXCL12 (10⁻⁸ M) or vehicle for 45 min and receptor surface expression was determined. Knockdown of β-arrestin1 and -2, 48 hrs after transfection, was assessed in western blot using an anti-β–arrestin1/2 antibody (inset). Anti-STAT3 (mAb 79D7, Cell Signaling Technologies) was used as loading control. (C) CXCR7 C-terminus is essential for receptor internalization. HEK293T cells were transfected with wt CXCR7 (filled bars), CXCR7 ΔC (grey bars) or CXCR7 ST/A (white bars) and cell surface receptor levels were assessed as above. Data represent the mean ± SEM of at least 3 experiments each performed in triplicate. ***, p<0.001 by one-way ANOVA and Bonferroni post test.