The SHSK motif prevents CXCR4 from constitutive down-modulation. (A,B) Effects of βarr2 on cell-surface expression of CXCR4^wt (A), CXCR4¹⁰¹³ (A), CXCR4^wt/Δi3 (B), and CXCR4^1013/Δi3 (B). HEK cells stably expressing either of the CXCR4 variants were transiently transfected with pN1-eGFP (N1) or βarr2-GFP. Expression of receptors without (□) or with treatment with 200 nM CXCL12 for 45 minutes at 37°C (■) was then assessed by flow cytometry. Results (means ± SD; n = 3) are receptor expression at the surface of GFP⁺-gated cells, expressed as percentage of the values in GFP⁺, pN1-eGFP–transfected cells in the absence of CXCL12 (100%). (C-F) The dot plots from flow cytometric assays represent cell-surface expression of CXCR4^wt (C), CXCR4¹⁰¹³ (D), CXCR4^wt/Δi3 (E), or CXCR4^1013/Δi3 (F) as a function of βarr2-GFP expression (ie, GFP fluorescence intensity) in transiently cotransfected HEK cells. Experiments (n = 3) were carried out 48 hours after transfection. (G) Fluorescence microscopic imaging of HEK cells stably expressing the GFP-tagged CXCR4 variant receptors.