Figure 4.

Syk is not required for phagocytosis of zymosan. Wild-type and Syk^-/- bone marrow-derived macrophages were cultured with fluorescently labeled zymosan for 15 minutes. Phagocytosis of zymosan was detected microscopically (A) and quantified by flow cytometry (B). (C) Bone marrow-derived macrophages were cultured with fluorescently labeled zymosan for the indicated times in the presence or absence of the Syk inhibitor piceatannol (25 μM) or the actin-disrupting agent cytochalasin D (2.5 μM). Phagocytosis was measured by flow cytometry, and the percentage of cells ingesting particles is shown. (D) HEK293 cells expressing GFP-tagged Dectin-1 were cultured with zymosan particles for 15 minutes, and Dectin-1 and actin were detected by immunofluorescence microscopy. (E) GFP/Dectin-expressing HEK293 cells were cultured with fluorescently labeled zymosan for 30 minutes in the presence or absence of cytochalasin D (E) or piceatannol (F), and internalization was measured by flow cytometry. (G) The cytoplasmic tail of murine Dectin-1 is depicted showing the 2 tyrosine residues and the triacidic motif. Residues that are conserved between mouse and human Dectin-1 are underlined. (H) HEK293 cells were transiently transfected with GFP-tagged Dectin-1 (wild type and the indicated mutants). The cells were cultured with fluorescently labeled zymosan, and internalization was measured by flow cytometry. The data are gated on GFP⁺ (Dectin-1-expressing) cells.