IgE(-Ag)–induced expression of Bcl family is impaired in IL-3^-/- BMMCs. (A) Expression of Bcl-xL protein on IgE(-Ag) stimulation in IL-3^+/+ or IL-3^-/- BMMCs. BMMCs were cultured with IL-3–depleted medium alone (None), 1 μg/mL IgE (IgE), or 10 ng/mL IL-3 (IL-3) for 24 hours. Cell lysates were analyzed by Western blotting with anti–Bcl-xL (upper panel) and anti-Lyn (lower panel) Abs as a control. (B) Expression of Bcl-xL and Bcl-2 on IgE(-Ag) stimulation in IL-3^+/+ or IL-3^-/- BMMCs. WT BMMCs (□) and IL-3^-/- BMMCs (▪) were cultured in the absence (none) or presence of 1 μg/mL IgE (IgE) for 2 hours. Relative mRNA levels of Bcl-xL and Bcl-2 were determined by real-time RT-PCR and expressed as fold induction over untreated cells after normalization with the values for GAPDH. (C) Bcl-xL expression is sufficient for inducing survival in IL-3^-/- BMMCs. Mature IL-3^-/- BMMCs were infected using pMX-IRES-GFP retroviral vector either alone (Mock) or together with Bcl-xL (Bcl-xL) as described in “Materials and methods.” ○ indicates mock/GFP-; ⬡, mock/GFP⁺; ▵, Bcl-xL/GFP^-; and ▴, Bcl-xL/GFP⁺. One day after infection, cells were cultured in the absence of IL-3 and cell viability analyzed for GFP⁺ or GFP^- negative population at the indicated periods. (D) STAT5 activation is sufficient for inducing mast cell survival. Mature WT BMMCs were infected with vector alone (Mock, □), wild-type STAT5a (STAT5a.WT, ▦), or constitutively active STAT5a (STAT5a.CA, ▪). Cell viability was analyzed for GFP⁺ (left panel) or GFP^- negative (right panel) population at indicated periods. (E) Indirect effect of active MEK on BMMC survival. WT BMMCs were infected with vector alone (Mock, □) or MEK.dSESE (active MEK, ▪). Cell viability was analyzed as described in panel D. (F) Expression of HDC mRNA on IgE(-Ag) stimulation in IL-3^+/+ (□) or IL-3^-/- (▪) BMMCs. The BMMCs were cultured as described in panel B. Relative mRNA expression was determined as indicated in panel B.