Dynamic localization of Kit and regulators of PI3K signaling after KL stimulation. (A,B) Kit is recruited to lipid rafts as a result of KL stimulation. (A) Subcellular fractions from unstimulated or KL-stimulated Mo7e cells (5 and 20 minutes), were isolated and immunoblotted with antibodies against Kit and PY. The signaling competent form of Kit and the tyrosine-phosphorylated protein comigrating with Kit are indicated by and , respectively. A protein of somewhat lower molecular mass cross-reacting with the anti-Kit antibody was observed in the cytosol and possibly represents a cytosolic Kit degradation product (⇨). For control of the fractionation procedure, subcellular fractions were also analyzed with antibodies against markers of the lipid raft (Flotillin-1), plasma membrane (Na/K ATPase), and cytosol (ERK2) fractions. (B) Verification of tyrosine-phosphorylated Kit in lipid rafts by IP. Because the data in A consist of an immunoblot that can only be used to infer that the approximately 160-kDa-phosphorylated band is Kit, an IP of lipid raft-localized Kit from resolubilized lysates was performed and analyzed with antibodies against Kit and PY. The coprecipitating, tyrosine-phosphorylated band of significantly higher molecular mass than Kit is of unknown origin. (C) Recruitment of p85 into lipid rafts, redistribution of the negative regulator PTEN from lipid rafts to the cytosol. Fractions were immunoblotted with antibodies against the p85 regulatory and p110 catalytic subunits of PI3K, PTEN, Akt and PDK1. (D) Complex formation between Kit and p85 in lipid rafts and verification of tyrosine phosphorylation of p85. As in B, an IP of Kit and tyrosine-phosphorylated proteins from lipid rafts was performed and analyzed with an antibody against p85.