FLNA repeats 19 and 23 bind to the CXCR4 ICL3 domain. (A) Pull-down assay of the indicated CXCR4 peptides with recombinant full-length FLNA protein. (B-C) ICL3 peptide pull-down assay with (B) purified His-tagged FLNA fragments or (C) individual FLNA repeats. Bound fragments or repeats were detected by immunoblot with peroxidase-labeled anti-His antibody. (D) Representative sensorgrams for FLNA repeat 19 binding kinetics to immobilized ICL3 peptide, monitored as relative units (RU). The concentrations of FLNA repeat 19 tested were 4 (violet), 2 (brown), 1 (red), 0.5 (blue), and 0.25 μM (green). Binding of FLNA repeat to the reference sensorchip (immobilized ICL3ΔCt) was subtracted for each experimental condition. Two sensorgrams are shown for each repeat 19 concentration. Langmuir fitting is shown in black. (E) Sensorgram for FLNA(16-23) binding kinetics to immobilized ICL3 peptide as in (D). The FLNA (16-23) concentrations tested were 155 (blue), 77.5 (green), 38.7 (brown), and 19.3 nM (cyan). The black line represents the fitting to the 1:1 model. H2-24, hinge region 2 + repeat 24; R1-R8, repeats 1-8.