The CXCR4 ICL3 domain bears an FLNA-binding motif. (A) Alignment of distinct FLNA partners, showing the consensus binding sequence; (*) indicates relevant amino acids. (B) Scheme of the CXCR4 peptides designed for pull-down assays and their position in the CXCR4 sequence. (C) Pull-down assay. Biotinylated peptides were incubated with HEK-293 cell lysates (Lys) and pull-down was performed. Immunoblot of total lysates (left) and peptide-bound proteins using anti-FLNA antibody (top right) and streptavidin (bottom right). A representative experiment is shown (n = 5). (D) Total lysates of HEK-293 cells expressing indicated GFP-fused receptors were immunoprecipitated (i.p.) with anti-FLNA antibody. Total lysates (left) and FLNA-bound proteins (right) were detected with anti-FLNA (top) and anti-GFP antibodies (bottom); arrowhead indicates the CXCR4 band. (E) Immunoblots as in (D) were quantified by densitometry, and the CXCR4:FLNA ratio was determined. Values were normalized to those for the CXCR4^S324X mutant, which has an unmodified ICL3 domain. Data are mean ± standard error of the mean (SEM; n = 3). **P < .01, 2-tailed Student t test. w.b., western blot.