Notch1

Western blot was performed as previously described [18 ]. To extract whole proteins, RIPA lysis buffer (150 mM NaCl, 50 mM Tris-hydrochloride [pH 7.5], 1% Nonidet, P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, and 1 mM sodium fluoride) were used. Protein concentrations of the samples were determined by Bradford protein assay (Bio-Rad, Hercules, CA, USA). An equal concentration of proteins was loaded and separated on SDS polyacrylamide gels by electrophoresis, then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) and blocked with 5% bovine serum albumin or skim milk in TBS-T. The blocked membranes were incubated with primary antibodies against Notch1 (Novus Biologicals, Littleton, CO, USA), Sox 2 (Abcam, Cambridge, UK), β-catenin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD44 (Cell Signaling Technology, Danvers, MA, USA), CD 133 (MACS, Bergisch Gladbach, Germany), and β-actin (Abcam) in a cold room overnight. After washing, membranes were incubated with secondary antibodies (Santa Cruz Biotechnology) for 1 h and visualized using an enhanced chemiluminescence reagent (Animal Genetics, Inc., Suwon, Korea).