Figure 4. Binding properties of CXCL12 and [3-NT⁷]CXCL12 to CXCR4 and ACKR3.

CXCL12 (•, filled circles) and [3-NT⁷]CXCL12 (□, open squares) were added (concentrations ranging from 3.75nM to 375nM) to CHO-CXCR4 cells (panel A) or CHO- ACKR3 cells (panel B), together with 12.5nM CXCL12^AF647 while the cells were kept on ice during the one hour incubation period. The results shown are the mean percentages (±SEM) of fluorescence compared to the control where only CXCL12^AF647 was added to the cells (n=8 for the CXCR4 experiments, n=4 for ACKR3). Statistical comparison between CXCL12 and [3-NT⁷]CXCL12 was performed using the Mann-Whitney U test. Panels C and D show FACS data from a representative experiment with CHO-CXCR4 and CHO-ACKR3, respectively. The remaining fluorescence of CXCL12^AF647 after competition with 37.5nM CXCL12 (black line) or 37.5nM [3-NT⁷]CXCL12 (red line) are represented by two almost completely overlaying curves. The histogram where only CXCL12^AF647 was added to the cell (grey area) shows the maximal fluorescence signal. The unstained control is also shown (dotted line).