Cross-linking of mLMIR5 induced the phosphorylation of several signaling molecules in mLMIR5-transduced mast cells, resulting in cytokine/chemokine production, cell adhesion, histamine release, and cell survival. (A) BMMCs transduced with mLMIR5 were stimulated with either control IgG or anti-mLMIR5 Ab for 3 or 15 minutes. Cell lysates were subjected to immunoblotting with either anti–phospho-p44/42 MAPK (pERK1/2), anti–phospho-p38 MAPK (pp38), or anti–phospho-Akt (pAkt) Ab. Equal loading was evaluated with by reprobing the immunoblots with Abs specific for ERK1/2, p38, or Akt. (B) BMMCs transduced with mLMIR5 were incubated with PBS, control IgG, or anti-mLMIR5 Ab for 12 hours. IL-6, TNF-α, and MCP-1 released into the culture supernatants were measured by ELISA. (C) BMMCs transduced with mLMIR5 in FN- or FB-coated plates were stimulated with PBS, control IgG, or anti-mLMIR5 Ab for 60 minutes. Adherent cells were measured as described in “Measurement of cytokines and histamines and adhesion assay.” (D) FLMCs transduced with mLMIR5 were incubated with PBS, control IgG, or anti-mLMIR5 Ab for 50 minutes. Alternatively, anti-TNP IgE-sensitized cells were incubated with TNP-BSA for 50 minutes. Histamine released in the culture supernatants was measured. (E) FLMCs transduced with mLMIR5 were incubated with either PBS, control IgG, anti-mLMIR5 Ab, or IgE (SPE-7) in the absence of IL-3. At indicated time points, cells were stained with PE-labeled annexin V to monitor apoptosis. Cells incubated in the presence of IL-3 were also analyzed. All data points correspond to the mean and the standard deviation (SD) of 4 independent experiments. Statistically significant differences are shown. *P < .05.