## 2 Results
### Binding of CXCL9(74-103) to Cellular GAGs Is Reduced by Adding Heparin or HS  
In the next step, we studied the effect of adding soluble GAGs in combination with TAMRA-labeled peptides to the CHO cells. To this end, one GAG (heparin, HS or HA) and one peptide (KKQK-3 mer, KKKQQK-2 mer or CXCL9(74-103)) were simultaneously added to either HS\({}^{+}\) or HS\({}^{-}\) CHO cells. Different doses of each GAG (0, 0.03, 0.1, 0.3, 1 and 3 \(\mu\)g)  
Figure 7: Peptides show differential binding capacity for cellular GAGs on CHO cells. TAMRA-labeled peptide (0.24 nmol; KAKAK-2 mer (gray), KKQK-3 mer (blue), KKKQQK-2 mer (green), CXCL9(74-103) (red), unstained (orange)) was added to either HS\({}^{+}\) or HS\({}^{-}\) CHO cells and binding was assessed using flow cytometry. (**A**,**B**) Histograms (of one representative experiment) of the HS\({}^{+}\) (**A**) and HS\({}^{-}\) (**B**) CHO cells are shown with the cell count on the y-axis and the fluorescence intensity of the TAMRA-labeled peptides on the x-axis. (**C**) Median fluorescence intensity (MFI) values were normalized first by subtracting the MFI of the unstained sample (background). The difference between the MFI of the HS\({}^{+}\) and the HS\({}^{-}\) CHO cells is displayed (y-axis) for each peptide (x-axis). (\(n\geq 7\); Mann–Whitney test, \(*\)\(p<0.05\), *****\(p<0.0001\) (comparison of the different peptides)). The amino acid sequences of the peptides are depicted on the right with TAMRA indicated in pink [\({}^{+}\)] and the GAG-binding motifs underlined.  
and one dose of the peptide (0.24 nmol) were used. The MFI values of the different TAMRA-labeled peptides that are bound to the HS\({}^{+}\) or HS\({}^{-}\) CHO cells are shown in Figure S2. The net MFI values were calculated by subtracting the MFI of the unstained sample from the MFI of the sample with TAMRA-labeled peptides, per cell type (HS\({}^{+}\) or HS\({}^{-}\) CHO cells). First, the binding of each peptide to either the HS\({}^{+}\) or HS\({}^{-}\) CHO cells was compared per dose of GAG. Although the absolute MFI values were not largely different for both the KKQK-3 mer and the KKKQQK-2 mer, a significantly higher amount of each peptide was bound to HS\({}^{+}\) CHO cells compared with HS\({}^{-}\) CHO cells (Figure S2). Second, the different doses of GAG were compared with the condition without GAG. This comparison was performed per GAG and per peptide. The MFI values of the TAMRA-labeled peptides bound to the HS\({}^{-}\) CHO cells were subtracted from those of the HS\({}^{+}\) CHO cells, per peptide and per (dose of) GAG. The resulting values (MFI [(HS\({}^{+}\)]-[HS\({}^{-}\)]); data not shown) were used for statistical analysis. A Kruskal-Wallis test showed no significant differences between the groups for the KKQK-3 mer (heparin, HS and HA; Figure S2A-C), the KKKQK-2 mer (heparin, HS and HA; Figure S2D-F) and CXCL9(74-103) (HA; Figure S2I). Hence, in these cases, the addition of soluble GAGs failed to reduce the binding of the peptides to the CHO cells. In contrast, a statistically significant reduction of binding was detected for CXCL9(74-103) with heparin (Figure S2G) and with HS (Figure S2H) (both \(p<0.0001\)). Subsequently, a Mann-Whitney test was applied to compare each individual dose of heparin and HS to the condition without GAG for CXCL9(74-103) (Figure S2G,H). Interestingly, the addition of heparin (the three highest doses: 0.3, 1 and 3 \(\mu\)g) significantly reduced the binding of CXCL9(74-103) to the CHO cells (Figure S2G). A similar result was observed for HS (Figure S2H), although only the two highest doses (1 and 3 \(\mu\)g) could significantly reduce the binding of the peptide to the cellular GAGs. As opposed to heparin and HS, the addition of HA did not affect the binding of CXCL9(74-103) to the cellular GAGs on the CHO cells (Figure S2I).