SCF mediates the association of IL-33R with c-Kit in dependency of its tyrosine kinase activity and the integrity of cholesterol-rich microdomains. (A) Wt BMMCs were stimulated with IL-33 or SCF as indicated, lysed, and subjected to c-Kit immunoprecipitation. Coprecipitated IL-33R was detected by immunoblot and quantified for 3 independent experiments. Results are shown as increase (mean ± SEM) relative to nonstimulated cells. Equal amounts of IL-33R in lysates were determined by immunoblotting (direct load). (B) Wt BMMCs were preincubated with imatinib and subsequently stimulated with SCF. Lysates were immunoprecipitated with an anti–c-Kit antibody and analyzed for coprecipitated IL-33R and IL-1RAcP. (C) Wt BMMCs were treated with the methyl-β-cyclodextrin (MβCD) for 2 hours and subsequently stimulated with either IL-33 or SCF. Lysates were immunoprecipitated with anti–c-Kit and analyzed for c-Kit, IL-33R, and IL1-RAcP. Phosphorylation of Y721–c-Kit and equal amounts of IL-33R were verified by immunoblot (direct load). (D) Wt BMMCs were left untreated or were treated with MβCD for 2 hours and subsequently stimulated with either IL-33, SCF, or both for 6 hours. Supernatants were analyzed for IL-6.