Phospo kit

Whole-cell protein lysates were prepared from parental and BYL719-resistant cell-monolayers using NP40 buffer containing protease inhibitors (Halt protease- and phosphatase-inhibitor cocktail; Thermo Fisher Scientific) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich). Proteins were separated on SDS-PAGE and transferred onto nitrocellulose membranes. Transfer efficiency was demonstrated by Ponceau S staining (Sigma-Aldrich). Membranes were blocked by 5% skimmed milk, followed by incubation at 4°C overnight with the presence of a primary antibody against KIT (A4502; Dako, Ely, UK), phospo-KIT (3391; Cell Signaling Technology, Leiden, Netherlands), AKT (9272; Cell Signaling Technology), phospo-AKT (9271; Cell Signaling Technology), MAPK (9102; Cell Signaling Technology), phospo-MAPK (9101; Cell Signaling Technology), mTOR (2972; Cell Signaling Technology), phospo-mTOR (2448; Cell Signaling Technology), PTEN (138G6; Cell Signaling Technology), and actin (A1978; Sigma-Aldrich). After rinsing, membranes were incubated with horseradish peroxidase–conjugated secondary antibody (Thermo Fisher Scientific) at room temperature for 2 hours. After further rinsing, immunoreactive bands were visualized by enhanced chemiluminescence (BioRad, Hercules, CA, USA) and signals captured and quantified using ChemiDoc (BioRad).