Internalization of cell-surface CD63 accompaniesS cerevisiaeyeast phagocytosis in immature DCs: association of CD63 with dectin-1. (A) A yeast phagocytosis assay was performed as described in “Materials and methods” in the presence or absence of α-mannan, laminarin, or both polyosides. The percentage of phagocytic DCs was calculated in triplicate (at least 200 cells were counted per point). The top panel shows an example of DCs bearing phagocytic vacuoles containing yeasts (left) and of DCs incubated in the presence of laminarin (right); objective lens: Plan-Neofluar 100 ×/1.3 NA, oil. The results shown are representative of at least 5 experiments. (B) CD63 surface expression was evaluated by FACS analysis after yeast phagocytosis in the presence or absence of α-mannan, laminarin, or both polyosides and in control DCs (not incubated with yeasts). Ordinate indicates number of positive cells and abscissa indicates MFI. The histograms shown are representative of at least 5 experiments. (C) Example of FC-5.01/CD63 complexes surrounding internalized yeasts as described in “Results.” Objective lens: Plan-Neofluar 100 ×/1.3 NA, oil. (D) Immunoprecipitation of CD63 was performed as described in “Materials and methods.” The immunoprecipitates were analyzed by 10% SDS-PAGE under nonreducing conditions and revealed with anti-dectin-1 polyclonal antibody followed by HRP-GAR and chemiluminescence. The antibody detected a double band of approximately 27 kDa and 23 kDa, which corresponds to the estimated molecular weight for the dectin-1 full-length and 1b isoforms.²⁵  Lane 1: DC extract. Lane 2: CD63 immunoprecipitate (IP 5.01). Lane 3: control immunoprecipitation performed using isotype-matched control IgG2a (IP IgG2a).