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Figure 4 | BMC Immunology

Figure 4

From: Carbon monoxide down-regulates α4β1 integrin-specific ligand binding and cell adhesion: a possible mechanism for cell mobilization

Figure 4

Effect of hemin on binding and dissociation of the LDV-FITC probe on resting and activated U937 cells. LDV-FITC probe binding and dissociation on U937 cells plotted as LDV-FITC fluorescence versus time. The data were normalized to the level of the non-specific signal determined by the addition of excess unlabeled competitor (LDV 2 μM), and therefore, no cell autofluorescence can be seen. A. The experiment involved sequential addition of the fluorescent LDV-FITC probe (25 nM), and different concentrations of hemin (6-100 μM) or DMSO (vehicle). The non-specific binding of the LDV-FITC probe was determined using excess unlabeled competitor (LDV). Ligand dissociation rates (koff) were determined by fitting the dissociation part of the curves (after LDV addition) to the single exponential equation. The range of koff is shown. B. The span of the single exponential fits for the dissociation curves (from panel A after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose-response fit (Hill slope = 1) was obtained using GraphPad Prism software. C. The experiment was conducted using U937 cells stably transfected with the FPR ΔST receptor, and involved sequential addition of the fluorescent LDV-FITC probe (4 nM), the high affinity FPR ligand N-formyl-Met-Leu-Phe-Phe (100 nM), hemin (1.5-100 μM) or DMSO (control), and LDV (2 μM). LDV-FITC dissociation rates (koff) were determined as described for panel A. D. The span of the single exponential fits for the dissociation curves (from panel C, after LDV addition) plotted versus logarithm of hemin concentration. Means ± SEM of two independent determinations are shown (n = 2). The sigmoidal dose-response fit was obtained analogously to panel B.

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