Cells were lysed in TNE buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM EDTA; 1% SDS) supplemented with protease inhibitor cocktail diluted 1:100 (Cytoskeleton Inc., Denver, CO) and 1 mM sodium orthovanadate. The lysates were sonicated on ice for 20 seconds. Protein concentration was measured using a bicinchoninic acid (BCA) assay kit (BCA Protein Assay Kit; Thermo Fisher Scientific/Pierce Protein Biology Products, Rockford, IL). Equivalent amounts of proteins were analyzed by SDS–polyacrylamide gel electrophoresis. After electrophoretic separation, proteins were transferred onto nitrocellulose membrane (Invitrogen) and incubated overnight at 4°C with the following antibodies: Jag2, 1:800 (#2210; Cell Signaling Technology, Danvers, MA), GAPDH, 1:5000 (#TRK5G4-6C5; RDI, Flanders, NJ), phospho-Erk1–2Thr202/Tyr204, phospho-AktSer473, Erk1–2, Akt (Cell Signaling Technology), all diluted 1:1000. Proteins were visualized with peroxidase-coupled secondary antibodies (KPL, Gaithersburg, MD), using enhanced chemiluminescence (ECL) for detection (PerkinElmer, Waltham, MA).