Figure 6
Figure 6. Cross-linking of human LMIR5 induced the activation via phosphorylation of Y188 in its cytoplasmic region in DAP12-deficient BMMCs. (A) WT or DAP12−/− BMMCs transduced with a Flag-tagged mLMIR5 were stimulated with control IgG, anti-mLMIR5 Ab, or PMA (top panels), while WT or DAP12−/− BMMCs transduced with Flag-tagged hLMIR5 were stimulated with control IgG, anti-Flag mAb, or PMA (bottom panels). IL-6 released into the culture supernatants was measured by ELISA. All data points correspond to the mean and the SD of 4 independent experiments. (B) DAP12−/− BMMCs transduced with either a Flag-tagged mLMIR5 or hLMIR5 were stimulated with the indicated Abs for 3 minutes. The amount of phosphorylated ERK1/2 was analyzed as described. (C) WT or DAP12−/− BMMCs transduced with Flag-tagged hLMIR5 or WT BMMCs transduced with Flag-tagged hLMIR5 (K158Q) were incubated with the indicated Abs. Immunoprecipitates of cell lysates with anti-Flag mAb were immunoblotted with anti-phosphotyrosine mAb (4G10) or anti-Flag mAb. Total cell lysates were also analyzed to detect the amount of phosphorylated ERK1/2. Vertical lines have been inserted to indicate a repositioned gel lane. (D) BMMCs transduced with Flag-tagged hLMIR5, hLMIR5 (Y188F), or mock were stimulated with the indicated Ab for 3 minutes or with 100 μm pervanadate (PV) for 10 minutes. Immunoprecipitates of cell lysates with anti-Flag mAb were blotted with antiphosphotyrosine mAb (4G10) or anti-Flag mAb. Vertical lines have been inserted to indicate a repositioned gel lane. (E-H) WT (E) or DAP12−/− (F) DAP10−/− (G), or DAP12−/−DAP10−/− (H) BMMCs transduced with Flag-tagged hLMIR5, hLMIR5 (K158Q), hLMIR5 (Y188F), or hLMIR5 (K158Q) (Y188F) were stimulated with control IgG or anti-Flag mAb. IL-6 released into the culture supernatants was measured by ELISA. All data points correspond to the mean and the SD of 3 independent experiments. K158Q, Y188F, or KQYF indicate hLMIR5 (K158Q), hLMIR5 (Y188F), or hLMIR5 (K158Q) (Y188F), respectively. (I) DAP12−/−DAP10−/− BMMCs transduced with Myc-tagged hLMIR5 (Y188F) were transfected with Flag-tagged DAP10, DAP12, or mock. Immunoprecipitates of cell lysates were immunoblotted with anti-Flag mAb (left panel). These cells were stimulated with control IgG or anti-Myc mAb. IL-6 released into the culture supernatants was measured by ELISA. All data points correspond to the mean and the SD of 3 independent experiments. Statistically significant differences are shown. *P < .05.

Cross-linking of human LMIR5 induced the activation via phosphorylation of Y188 in its cytoplasmic region in DAP12-deficient BMMCs. (A) WT or DAP12−/− BMMCs transduced with a Flag-tagged mLMIR5 were stimulated with control IgG, anti-mLMIR5 Ab, or PMA (top panels), while WT or DAP12−/− BMMCs transduced with Flag-tagged hLMIR5 were stimulated with control IgG, anti-Flag mAb, or PMA (bottom panels). IL-6 released into the culture supernatants was measured by ELISA. All data points correspond to the mean and the SD of 4 independent experiments. (B) DAP12−/− BMMCs transduced with either a Flag-tagged mLMIR5 or hLMIR5 were stimulated with the indicated Abs for 3 minutes. The amount of phosphorylated ERK1/2 was analyzed as described. (C) WT or DAP12−/− BMMCs transduced with Flag-tagged hLMIR5 or WT BMMCs transduced with Flag-tagged hLMIR5 (K158Q) were incubated with the indicated Abs. Immunoprecipitates of cell lysates with anti-Flag mAb were immunoblotted with anti-phosphotyrosine mAb (4G10) or anti-Flag mAb. Total cell lysates were also analyzed to detect the amount of phosphorylated ERK1/2. Vertical lines have been inserted to indicate a repositioned gel lane. (D) BMMCs transduced with Flag-tagged hLMIR5, hLMIR5 (Y188F), or mock were stimulated with the indicated Ab for 3 minutes or with 100 μm pervanadate (PV) for 10 minutes. Immunoprecipitates of cell lysates with anti-Flag mAb were blotted with antiphosphotyrosine mAb (4G10) or anti-Flag mAb. Vertical lines have been inserted to indicate a repositioned gel lane. (E-H) WT (E) or DAP12−/− (F) DAP10−/− (G), or DAP12−/−DAP10−/− (H) BMMCs transduced with Flag-tagged hLMIR5, hLMIR5 (K158Q), hLMIR5 (Y188F), or hLMIR5 (K158Q) (Y188F) were stimulated with control IgG or anti-Flag mAb. IL-6 released into the culture supernatants was measured by ELISA. All data points correspond to the mean and the SD of 3 independent experiments. K158Q, Y188F, or KQYF indicate hLMIR5 (K158Q), hLMIR5 (Y188F), or hLMIR5 (K158Q) (Y188F), respectively. (I) DAP12−/−DAP10−/− BMMCs transduced with Myc-tagged hLMIR5 (Y188F) were transfected with Flag-tagged DAP10, DAP12, or mock. Immunoprecipitates of cell lysates were immunoblotted with anti-Flag mAb (left panel). These cells were stimulated with control IgG or anti-Myc mAb. IL-6 released into the culture supernatants was measured by ELISA. All data points correspond to the mean and the SD of 3 independent experiments. Statistically significant differences are shown. *P < .05.

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