Anti jagged1

Cellular proteins were extracted by using Nonidet-P40 lysate buffer (1% NP40, 200 nM NaCl, 50 mM Tris pH 7.4) with addition of protease and phosphatase inhibitors (Sigma-Aldrich Inc.), maintained on ice for 30 min, vortexed and then centrifuged at 12000 rpm for 10 min at 4°C. Protein concentration was determined using BioRad protein-assay (Bio-Rad Laboratories, Inc.). Analysis of protein expression was performed using capillary WES™ technology by ProteinSimple (Bio-Techne, Minneapolis, MN, USA) according to the manufacturer’s protocol. 500 ng of each sample were added to the fluorescent 5XMaster Mix and loaded into a Wes 12–230 kDa plate, together with a biotinylated ladder, primary antibodies, ready-to-use HRP-conjugated anti-rabbit or anti-mouse secondary antibody and luminol-peroxide mix. The acquisition and quantitative analysis of images were executed with Compass software (ProteinSimple). The following primary antibodies were used: 1:25 anti-Jagged-1 (#2620, Cell Signaling, Danvers, MA, USA), 1:30 anti-pAKT(Ser473)(#4058, Cell Signaling), 1:75 anti-AKT (#9272, Cell Signaling) and 1:100 anti-β-actin (A5441, Sigma-Aldrich Inc.).