Figure 3.

Dectin-1 signaling is sufficient to activate Syk in a subpopulation of macrophages. (A) Expression of SBP-tagged Dectin-1 in a stably transfected population of RAW264.7 macrophages was assessed by flow cytometry using an antibody to the epitope tag. (B) SBP-tagged Dectin-1 was localized by immunofluorescence microscopy in resting cells (left panel) or in cells fed streptavidin-coated beads (right panel). (C) RAW264.7 cells were incubated with streptavidin-coated beads in the presence of cytochalasin D (2.5 μM) or biotin (100 μM) as indicated, and internalization of the particles was measured by flow cytometry. (D) RAW264.7 cells expressing SBP-tagged Dectin-1 were incubated with streptavidin-coated beads (left panel) or zymosan (right panel) in the presence of laminarin (0.5 mg/mL) or biotin (100 μM) as indicated. ROS production was measured by luminol-enhanced chemiluminescence. (E) RAW264.7 cells expressing SBP-tagged Dectin-1 were stimulated with streptavidin-coated beads for 20 minutes in the presence of sodium orthovanadate, and activation of Syk was visualized by immunofluorescence microscopy as in Figure 1. (F) Activation of Syk in RAW264.7 cells expressing SBP-tagged Dectin-1 and stimulated with streptavidin-coated beads was assessed by flow cytometry.