Ligand-induced internalization and recycling in presence or absence of receptor antagonists.

<p>A: RBL cells stably expressing CXCR4- (left) or CCR5-AP (right) were enzymatically biotinylated and incubated in BM medium containing 125 nM ligand (30’/37°C). Recycling was triggered by acid wash to remove the ligand and transfer into ligand-free medium (30’/37°C). Cells were stained with anti-AP (dashed line) or streptavidin (straight line) and analyzed by flow cytometry. Each curve shows the mean percentage (+/- s.d.) of receptors expressed on the cell surface normalized to the MCF value of untreated cells. B: Cells were treated and stained accordingly to panel A (grey bars anti-AP, black bars streptavidin). During the recycling phase the corresponding antagonist was added (TAK779 3 μM; AMD3100 30 μM). C: RBL cells expressing CCR5-AP S/A (filled triangle up) or CCR5-AP WT (open square) were enzymatically biotinylated. Internalization was triggered as described in panel A. Staining was done with streptavidin-PE. Receptor recycling was calculated as percentage of the difference between cell surface expression of the receptor at time points 0 and 30 minutes Results represent mean +/- s.d. of at least three independent experiments. n.s., not significant; **, p < 0.001).