Journal: PLoS ONE

Article Title: Ligand Binding and Signaling of Dendritic Cell Immunoreceptor (DCIR) Is Modulated by the Glycosylation of the Carbohydrate Recognition Domain

doi: 10.1371/journal.pone.0066266

Figure Lengend Snippet: (A) Schematic overview of the N -glycosylation site inside the putative CRD of DCIR. NES is the only predicted N -glycosylation site of DCIR. The EPS motif is the predicted glycan binding motif. Adapted from Bates et al . . (B) Increased binding to DCIR ligands in the presence of truncated or complete absence of glycans on the DCIR-Fc construct. Binding of wild-type and the glycosylation mutant DCIR-Fc to immobilized neoglycoconjugates was measured in a glycan binding assay. Average DCIR-Fc binding ± SEM was calculated from 4 independent experiments. (C) DCIR expression on DCIR transduced cell lines was determined by flow cytometry. Filled histogram represents isotype control and black line indicates the DCIR expression. (D) Binding of glycan beads to cellular DCIR could not be detected. Binding of fluorescent labeled glycan beads to the different DCIR expressing cell lines was measured by flow cytometry in the absence of serum. As a control binding of the fluorescent labeled glycan beads to CHO-DC-SIGN was measured. One representative experiment out of 3 is shown. (E) Glycan binding of PAA-glycans to cellular DCIR could not be detected. Binding of biotinylated PAA-glycans pre-incubated with streptavidin-Alexa Fluor 647 to the different DCIR and DC-SIGN expressing cell lines was measured by flow cytometry after 2 hours incubation at 37°C. Binding of biotinylated PAA-glycans to CHO-DC-SIGN served as a positive control. One representative experiment out of 3 is shown.

Article Snippet: Antibodies used are α-DCIR 111F8.04 (Dendritics), α-DC-SIGN AZN-D1 , α-Langerin 10E2 , α-phosporylated tyrosine PY20 (Millipore), goat anti-mouse IgG Alexa Fluor 488 F(ab′) 2 fragment (Invitrogen), goat-anti-human Fc-PO (Jackson Immunoresearch), strepatavidin-PO (Invitrogen), goat-anti-human Fc (Jackson), goat-anti-human Fc-biotin (Jackson), streptavidin-Alexa Fluor 647 and 488 (Invitrogen) and IRDye goat-α-mouse IgG 800CW (Li-Cor).

Techniques: Binding Assay, Construct, Mutagenesis, Expressing, Flow Cytometry, Labeling, Incubation, Positive Control