FIG 5.
Dectin-1 activates Syk and produces ROS to induce the expression of PIAS-1 and SOCS-1. (A and B) Immunoblot analysis of pSyk (A) and detection of ROS by DCFDA (B) in mouse peritoneal macrophages treated with curdlan at the indicated time points. −, no curdlan treatment. (C and D) RAW 264.7 macrophages are transiently transfected with Dectin-1 siRNA followed by treatment with curdlan for 60 min. Immunoblotting analysis of pSyk in whole-cell lysates (C) and ROS production (D) was analyzed by DCFDA. (E) Mouse peritoneal macrophages were pretreated with Syk inhibitor for 60 min and then treated with curdlan for 12 h. Total cell lysates were analyzed for PIAS-1 and SOCS-1 by immunoblotting. (F) ROS detection in macrophages from WT or TLR2−/− mice which are treated with curdlan for 120 min. (G) Peritoneal macrophages isolated from C57BL/6J WT and NCF1−/− mice were treated with curdlan for 12 h. Lysates were analyzed for PIAS-1 and SOCS-1 by immunoblotting. (H) C57BL/6J WT or NCF1−/− mice were intravenously challenged with curdlan for 12 h. Spleen and lymph nodes were isolated and analyzed for PIAS1 and SOCS-1 expression by quantitative real-time RT-PCR. All blots are representative of 3 independent experiments, and all data represent the means ± SEM from 3 independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001; Med, medium; WT, wild type; NCF1−/−, p47phox−/− (mice deficient in ROS production).