Phe-29, Phe-189, and Lys-271 of CXCR4 are important for ubiquitin binding but do not contribute to the binding of SDF-1α. A, FACS analyses of FITC-ubiquitin binding (1 min, 4 °C) after transfection of HEK293 cells as in Fig. 4. Top, 1 μg/ml FITC-ubiquitin. Bottom, 10 μg/ml FITC-ubiquitin. Black, cells transfected with empty vector. Red, cells transfected with HA-tagged WT CXCR4. Blue, cells transfected with HA-tagged CXCR4-F29A. Green, cells transfected with HA-tagged CXCR4-F189A. Light green, cells transfected with HA-tagged CXCR4-K271A. RFU, relative fluorescence units. B, FACS analyses of FITC-SDF-1α binding (1 min, 4 °C) after transfection of HEK293 cells as in Fig. 4. Top, 1 μg/ml FITC-SDF-1α. Bottom, 10 μg/ml FITC-SDF-1α. Same color code as in A. C and D, quantification of the mean fluorescence signals from three experiments as in A and B. C, 1 μg/ml FITC-ubiquitin/SDF-1α. D, 10 μg/ml FITC-ubiquitin/SDF-1α. Data are expressed as the percentage of binding to WT CXCR4.