CXCL12 enhances CXCR4-FLNA interaction in a ROCK- and ICL3-dependent manner. (A) X4^WT- or X4-ICL3ΔCt–expressing HEK-293 cells were pretreated with the ROCK inhibitor Y27632 or vehicle (Vhcl; dimethylsulfoxide), then left untreated or stimulated for 10 or 20 minutes with CXCL12. Cell lysates were immunoprecipitated with anti-GFP (CXCR4 tag) antibody and immunoblotted with anti-FLNA (top) and anti-GFP (bottom) antibodies. The relative ratio is shown for CXCR4:FLNA (FLNA), calculated by densitometric analysis (right); values were normalized to the ratio in unstimulated cells. (B) HEK-293 cell were pretreated as in (B) and stimulated with CXCL12 for 5 minutes or unstimulated. Cell lysates were immunoprecipitated with anti-FLNA, and resolved proteins were blotted with anti-FLNA and anti-GFP antibodies. The relative CXCR4:FLNA ratio was calculated as above. (A-B) Data are mean ± SEM (n = 3). **P < .01; ***P < .001; 2-tailed Student t test.