Recombinant expression and functional analysis of the hDectin-1b ECD. The cloned ECD of hDectin-1b was recombinantly expressed in E coli, purified, and subjected to a renaturation procedure. (A) Purity of the protein was analyzed in SDS-PAGE following silver staining. (B-D) Analysis of the binding behavior of the ECD. Shown are FACS stainings with (open histogram) or without (solid histogram) the ECD of hDectin-1. Detection was done using an antibody that recognizes the 6xHis tag of the ECD followed by incubation with an appropriate FITC-conjugated secondary antibody. (B) Analysis of the binding of the ECD to different tumor cell lines. (C) Specific binding of the hDectin-1b ECD was verified by precipitating the recombinant protein with macromolecular zymosan particles prior to the incubation of the staining solution with K562 cells. (D) Control staining with DHFR, an irrelevant protein produced in an analogous manner as the ECD. (E) To characterize the ligand for hDectin-1 on K562, cells were treated for 10 minutes with N-glycosidase (10.0 U/mL), pronase (5.0 mg/mL), or proteinase K (5.0 mg/mL) or were left untreated. The percentage of positive cells (B,D,E) or the mean fluorescence intensity (E) is shown in the upper right corner of the histograms. The data shown are representative of at least 3 independent experiments.