ICL3-FLNA interaction regulates CXCL12-induced internalization of WHIM-like CXCR4 receptors. (A) FACS analysis of cell surface expression of X4^WT, X4-ICL3ΔNt, and X4-ICL3ΔCt in unstimulated HEK-293 cells; a representative experiment is shown (n = 4). (B) Kinetics of X4^WT, X4-ICL3ΔNt, and X4-ICL3ΔCt internalization in HEK-293 cells after CXCL12 stimulation. Cell surface expression was determined with an anti-CXCR4 antibody in the GFP⁺ cell population and calculated as indicated (see “Methods”). (C) HEK-293 cells were transfected with X4^WHIM, X4^WHIM-ICL3ΔNt, and X4^WHIM-ICL3ΔCt, and CXCL12-induced internalization kinetics were analyzed as above. (D) Representative histograms of cell surface levels of X4^WHIM, X4^WHIM-ICL3ΔNt, and X4^WHIM-ICL3ΔCt before and after 60 minutes of CXCL12 stimulation. (B-C) n = 4. ***P < .001, 2-way analysis of variance (ANOVA) with Bonferroni posttest. (E) HEK-293T cells were transiently transfected with GFP-tagged X4^WHIM and X4^WHIM-ICL3ΔCt (green), and colocalization with the EEA1 (red) was analyzed by immunofluorescence (a,c) before and (b,d) after 5 minutes of CXCL12 stimulation. (F) Colocalization of the aforementioned receptors (green) with LAMP1 (red) in (a,c) resting and (b,d) CXCL12 cells stimulated for 15 minutes. (E-F) Cells shown are representative of at least 10 fields; bar = 10 μm.