Figure 2

Effect of nitric oxide addition on binding and dissociation of the LDV-FITC probe on U937 cells, treated with different Gα _i -coupled receptor ligands. LDV-FITC probe binding and dissociation on U937 cells stably transfected with different GPCRs plotted as mean channel fluorescence (MCF) versus time. A, The experiment involved sequential addition of fluorescent LDV-FITC probe (4 nM, below saturation, added 2 min prior to addition of Gαi-coupled receptor ligand, fMLFF, 100 nM), and different concentrations of DEA-NONOate (nitric oxide donor). Dashed line indicates the non-specific binding of the LDV-FITC probe determined using an excess of unlabelled LDV competitor (as shown in Fig. 2D,E). B, The experiment involved sequential addition of fluorescent LDV-FITC probe (4 nM), CXCL12/SDF-1 (12 nM), and DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control). Rapid and reversible binding of the probe reflects the VLA-4 affinity change [14]. C, The experiment involved sequential addition of the fluorescent LDV-FITC probe (4 nM), CXCL8/IL-8 (20 nM), and DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control). D, The experiment involved sequential addition of the DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), CXCL12/SDF-1 (12 nM). Excess unlabelled competitor LDV (1 μM) is added at the end of the experiment to determine the non-specific binding of the probe (panels D, and E). E, The experiment involved sequential addition of DEA-NONOate (250 μM, nitric oxide donor) or vehicle (control) at the 0 time point, and the fluorescent LDV-FITC probe (4 nM), CXCL8/IL-8 (20 nM) (arrows). According to the unpaired t test, the means are significantly different (p<0.05) at the peak of activation (marked on panels D and E as “*”), and at the steady state (marked on panels B-E as “**”). Experiments shown in the different panels were performed using different instruments, and therefore MCF values are not identical.