Figure 3. Binding of ¹²⁵I-labeled IL-8 to COS-7 cells expressing WT CXCR1 or its mutants.

The CXCR1 mutants were transiently transfected in COS-7 cells. Maximal binding of ¹²⁵I-IL-8 to CXCR1 WT and mutants (A) and competition binding experiments (B and C) were performed as described under “Materials and Methods”. Competitive binding studies were conducted using ¹²⁵I-labeled IL-8 and various unlabelled ligands as described under “Materials and Methods”. Representative maximal binding experiments for L128 and V247 mutants are shown in Fig. 3A, performed in triplicates. Mock was transfected with pSG5 plasmid. Representative competitive binding experiments for L128 and V247 mutants are shown in Fig. 3(B) and Fig. 3(C), performed in triplicates. Nonspecific binding was determined by adding 250 nM unlabeled IL-8. Curve fitting was done using GraphPad Prism data analysis program, and the affinity constant (K_d) for CXCR1 mutants is shown in Table 1.