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. 2012 Mar 12;209(3):551–563. doi: 10.1084/jem.20111739

Figure 5.

Figure 5.

CXCL12 and the CXCL12–HMGB1 heterocomplex trigger different conformational changes in CXCR4 homodimers. (A and B) FRET saturation curves were generated in HEK293T cells transiently cotransfected with a constant amount of CXCR4-CFP (CXCR4-C; 2.0 µg; ∼300,000 FU) and increasing quantities of CXCR4-YFP (CXCR4-Y; 0.25–4.25 µg; ∼80,000-2,000,000 FU) or mGluR1α-Y (0.5–6.0 µg; ∼110,000-2,100,000 FU) as negative control. The curves represent data obtained in eight independent experiments. (B) Effect of CXCL12 (100 nM, 30 min) or CXCL12 + HMGB1 (100 nM and 300 nM, 30 min) on CXCR4 homodimers. The curves represent data obtained in 12 independent experiments. (C) FRETmax and FRET50 values shown were deduced using a nonlinear regression equation applied to a single binding site model and are representative of 8–12 independent experiments. FRETmax signals for CXCL12-treated and CXCL12–HMGB1-treated cells increase significantly compared with untreated cells (*, P < 0.05). Furthermore, the FRETmax value for CXCL12-treated cells is significantly higher (*, P < 0.05) than the FRETmax for HMGB1–CXCL12-treated cells. Error bars indicate SEM.