SDF-1 affinity for heparin and cell surface proteoglycans. (A) Recombinant full-length SDF-1β (1-72) and SDF-1α (1-68) and synthetic SDF-1α 1-67 and 3-67 were injected at various concentrations (200, 100, 50, 25, and 12.5 nM) over a Biacore sensor chip containing streptavidin plus biotinylated heparin. The signal (measured in resonance units [RUs]) was recorded over a 120-second association phase and 120-second dissociation phase. The dissociation equilibrium constants (K_d) are shown for each set of data. Representative results from 3 independent experiments performed are shown. (B) SDF-1α binding to HUVECs evaluated by fluorescence-activated cell sorter (FACS) analysis. Recombinant full-length SDF-1α (1-68) and synthetic SDF-1α 1-67 were added at various concentrations (2000, 1000, and 500 ng) to HUVECs (1 × 10⁶ cells). After incubation (4°C, 30 minutes), the cells were stained for surface SDF-1. A representative experiment of 3 performed is shown. Filled histograms reflect background fluorescence. (C) Recombinant SDF-1α (50 ng) was incubated for 10 minutes in buffer alone or with heparin (50 or 1000 μg/mL) with or without addition of fresh human serum. Lane 1: SDF-1α alone; lane 2: SDF-1α plus 10% human serum; lane 3: SDF-1α plus heparin 50 μg/mL; lane 4: SDF-1α plus heparin 1000 μg/mL; lane 5: SDF-1α plus heparin 50 μg/mL plus 10% human serum; lane 6: SDF-1α plus heparin 1000 μg/mL plus 10% human serum. Samples were immunoblotted with rabbit (top) or goat (bottom) antibodies against SDF-1α and SDF-1β.