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. 2011 Oct 28;286(51):44145–44152. doi: 10.1074/jbc.M111.298505

FIGURE 4.

FIGURE 4.

Phe-29, Phe-189, and Lys-271 of CXCR4 are important for ubiquitin binding. A, HA-tagged open reading frame cDNA clones of WT CXCR4, CXCR4-F29A, CXCR4-F-189A, and CXCR4-K271A were transfected into HEK293 cells followed by immunoblotting of whole cell lysates with anti-HA and anti-GAPDH. EV, empty vector. B, quantification of HA expression by flow cytometry after transfection as in A. Thick lines, cells labeled with mouse anti-HA/anti-mouse Alexa Fluor 488 goat IgG. Thin lines, control, cells labeled with rabbit IgG/anti-rabbit FITC goat IgG. Gray, unstained cells. 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. C, FITC-ubiquitin binding (1 min, 4 °C) after transfection as in A and B. White circle, empty vector. Large black circle, WT CXCR4. White square, CXCR4-F29A. White upright triangle, CXCR4-F189A. White inverted triangle, CXCR4-K271A. Small black circle, nonspecific binding was similar after transfection with empty vector and the various CXCR4 cDNA clones. D, quantification of Bmax from five independent experiments as in C.