Figure 3. Mincle-mediated cellular signaling through Syk in RBL-2H3 cells.

(a) Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc) or preincubated overnight with anti-DNP IgE mAb and then stimulated with 300 ng/ml DNP-BSA for 10 min (DNP). Detergent-soluble lysates were reacted with GST-Syk-SH2 prebound to glutathione Sepharose 4B beads. The bound proteins were analyzed by immunoblotting with the indicated antibodies. Arrowheads show the positions of FcεRIβ and FcεRIγ. (b) Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without 10 μg/ml anti-myc mAb for 30 min. Detergent-soluble lysates were immunoprecipitated with anti-phosphotyrosine (pY) mAb-conjugated agarose beads, and then the sources of precipitation (input) and immunoprecipitates (IP) were analyzed by immunoblotting with the indicated antibodies. Arrowheads show the position of Syk. (c and d) Cell lines expressing WT Mincle were preincubated with the indicated concentrations of R406 or BAY61-3606 for 5 min, and then stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc) or preincubated overnight with anti-DNP IgE mAb and then stimulated with 300 ng/ml DNP-BSA for 10 min (DNP). (c) Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. (d) Detergent-soluble lysates were immunoprecipitated with the anti-PLCγ2 antibody, and then immunoprecipitates (IP) and the sources of precipitation (input) were analyzed by immunoblotting with the indicated antibodies. (e) Cell lines expressing WT Mincle or the R42I mutant were stimulated with or without plate-coated TDM for 60 min. Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. (a–e), Molecular size markers are indicated at the left in kDa. Data are representative of three independent experiments using PA-11 (WT) and R42I-3 (R42I) cell lines. Similar results were obtained from the other cloned cell lines.