Figure 5. CXCR4 internalization and β-arrestin 2 recruitment through CXCR4 and ACKR3 in response to CXCL12 and [3-NT⁷]CXCL12 stimulation.

CXCL12 and its nitrated counterpart were compared for their ability to induce CXCR4 internalization (panel A) and β-arrestin 2 recruitment (panel B) through activation of CXCR4 or ACKR3. A. Using specific anti-CXCR4 antibodies, remaining receptor expression on CHO-CXCR4 cells was tested after one hour incubation at 37°C with CXCL12 (•, filled circles) or [3-NT⁷]CXCL12 (□, open squares). Both forms were added at concentrations ranging from 3.75 to 375nM. For each experiment, the fluorescence measured after stimulation with buffer only was set to 100%. Statistical analysis (n=4; ± SEM) was performed using the Mann-Whitney U test (* p < 0.05). B. The recruitment of β-arrestin 2 in function of the ligand concentration (0.01nM to 1μM) was compared for CXCL12 (•, filled circles) and [3-NT⁷]CXCL12 (□, open squares) in CXCR4-transfected C2C12 cells (n ≥ 4) or ACKR3-transfected U2OS cells (n=3). The data shown are a mean percentage (± SEM) of the maximal β-arrestin 2 recruitment with unmodified CXCL12. Statistical differences between the two CXCL12 forms were analyzed using the Mann-Whitney U test.