Prestimulation of wt BMMCs with SCF is crucial for the full biologic function of IL-33. (A) Wt BMMCs were stimulated with SCF or IL-33 as indicated. Costimulation was performed by prestimulation with SCF for 30 minutes followed by stimulation with IL-33 for additional 30 minutes. Lysates were analyzed by immunoblot for phosphorylated signaling molecules as indicated. (B) Wt or kit^W-sh/W-sh BMMCs were stimulated with SCF and/or IL-33 for 24 hours. Supernatants were analyzed for IL-6. (C-D) Wt BMMCs were preincubated with imatinib for 30 minutes and subsequently prestimulated with SCF for 30 minutes followed by stimulation with IL-33 for additional 30 minutes (C) or 24 hours (D). Lysates were analyzed by immunoblot for phosphorylated signaling molecules as indicated. Phosphorylation was quantified in 3 independent experiments and results are shown as increase (mean ± SEM) relative to nonstimulated cells (C). Supernatants were analyzed for IL-6 (D). (E) Wt BMMCs were stimulated with SCF and/or IL-1β or IL-33 for 24 hours. Supernatants were analyzed for IL-6.