Fig. 4. The interactions of EPI-X4 and the EPI-X4 derivatives WSC02 and JM#21 with point-mutated CXCR4 were assessed by an antibody competition assay.

Amino acid substitutions were introduced in the sequence of CXCR4 by site-directed mutagenesis, cloned into an IRES-eGFP expression vector, and transfected into 293 T cells. Afterward, cells were incubated with serially diluted EPI-X4 (a, b), WSC02 (c, d), or JM#21 (e, f) in presence of a constant concentration of CXCR4 specific antibody (clone 12G5). After 2 h, bound antibody was analyzed by flow cytometry. a, c, e) Representative dose-dependent replacement of 12G5 antibody by peptides. b, d, f) IC₅₀ values were calculated by nonlinear regression. Shown are data derived from at least three individual experiments ± SEM. IC₅₀ values were set to 1000 µM (EPI-X4 and WSC02) or 100 µM (JM#21) if the value exceeds the maximally used concentration. *p ≤ 0.05, ***p ≤ 0.001 (one-way ANOVA for comparison with wt). For IC₅₀ values see Table 1. For all binding curves see also Fig. S8.