The intracellular trafficking of Dectin-1 depends on the nature of the β-glucan ligand. After overnight incubation, thioglycollate-elicited macrophages were pretreated with or without cycloheximide (cyclo) and then incubated with an excess of unlabeled (A) zymosan (zym) or (B) laminarin (lam) or glucan phosphate (gluP) for 60 minutes at 4°C. Cells were then incubated at 37°C for the times indicated, before the extent of Dectin-1 recovery at the cell surface was measured using cold FITC-labeled zymosan. The results are expressed as the percentage recovery of Dectin-1 versus control cells, at 160 minutes, that were not exposed to carbohydrate. Values are expressed as the mean ± SEM from triplicate samples and are representative of at least 3 independent experiments. Similar receptor kinetics were also observed by FACS analysis using 2A11 (data not shown). (C) To confirm that the recovered zymosan-binding capacity was due to Dectin-1, control cells from the final time point were treated with monoclonal antibody 2A11 to block the Dectin-1 lectin-binding site for 60 minutes prior to adding the FITC zymosan particles. The contribution of Dectin-1 was also confirmed in cells pretreated with zymosan, laminarin, and glucan phosphate (data not shown).