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. 2017 Apr 10;7:46064. doi: 10.1038/srep46064

Figure 4. Engagement of Mincle induces Syk- and PLCγ2-dependent activation of ERK and NFAT.

Figure 4

(a) Syk-deficient cells (Syk KO) and PLCγ2-deficient cells (PLCγ2 KO) were established from cells expressing WT Mincle (WT) by the CRISPR/Cas9 system with corresponding gRNAs. The cells were stimulated with or without 10 μg/ml anti-myc mAb for 30 min (anti-myc). Detergent-soluble lysates were analyzed by immunoblotting with the indicated antibodies. Molecular size markers are indicated at the left in kDa. (b) Cells expressing WT Mincle (WT), Syk-deficient cells (Syk KO), and PLCγ2-deficient cells (PLCγ2 KO) were transiently transfected with luciferase reporter plasmids. At 24 h after transfection, the cells were stimulated with or without 10 μg/ml anti-myc mAb for 6 h (anti-myc). As a control, the transfected cells were preincubated with anti-DNP IgE mAb and then stimulated with 30 ng/ml DNP-BSA for 6 h (DNP). Normalized luciferase activities are expressed as -fold of increase compared with unstimulated cells. Data are presented as the mean ± S.D. (*P < 0.01 versus WT Mincle-expressing PA-11 cells stimulated with the anti-myc mAb and **P < 0.05 versus WT Mincle-expressing PA-11 cells stimulated with DNP-BSA were considered significant. n = 3/group). (a and b) Data are representative of three independent experiments using gRNA#1-derived Syk- and PLCγ2-deficient cells. Similar results were obtained from the other cloned cell lines as well as gRNA#2-derived knockout cells. (c) Cells expressing WT Mincle (WT) and Syk-deficient cells (Syk KO) were stimulated with or without 10 μg/ml anti-myc mAb (anti-myc) for 20 min, or preincubated overnight with anti-DNP IgE mAb and then stimulated with 30 ng/ml DNP-BSA for 20 min (DNP). Detergent-soluble lysates of cytoplasmic (Cytosol) and nuclear cell fractions (Nucleus) were analyzed by immunoblotting with the indicated antibodies. Arrowheads show the positions of NFAT family proteins. Molecular size markers are indicated at the left in kDa. Data are representative of three independent experiments using PA-11 (WT) and gRNA#1-derived Syk-deficient cells. Similar results were obtained from the other cloned cell lines.