Binding and dissociation of the LDV-FITC probe in response to Gα
-coupled receptor agonist. Experiments were conducted as described under "Methods". A, LDV-FITC probe binding and dissociation on peripheral blood monocytes plotted as mean channel fluorescence (MCF) versus time. The experiment analogous to the one shown in Fig. 1A involved sequential additions of Gαs-coupled receptor ligand (isoproterenol (100 nM)) or DMSO (control), next, fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of Gαi-coupled receptor ligand). Cells were activated with A, SDF-1 (25 nM, arrow), or B, fMLFF (100 nM, arrow). Rapid and reversible binding of the probe reflects a transient VLA-4 affinity change. Curves are means out of four independent runs calculated on a point-by-point basis. One representative experiment out of two independent experiments for each ligand is shown. Experiments shown in the different panels were performed using different instruments, and therefore MCF values are not identical.