Lysis buffer (1% Triton X-100 (Sigma-Aldrich), 100 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10% glycerol (Amresco, Solon OH, USA), 50 mM sodium fluoride (Sigma-Aldrich), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), 1 mM p-nitrophenyl phosphate (Sigma-Aldrich), and 1 mM sodium orthovanadate (Sigma-Aldrich)) was used to lyse the cells, and the cell lysates were centrifuged at 13,000 rpm for 15 min at 4 °C. Bradford protein assay reagent (Bio-Rad) was used to quantify the amount of protein in the supernatant, and 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved these proteins. The separated proteins were then placed on a nitrocellulose paper membrane (Bio-Rad), where they were blocked with 5% skimmed milk in Tris-buffered saline for 1 h. Following that, the appropriate primary antibodies were used to incubate the membranes against GPR50 (14032S, 1:1000), NANOG (SC33759, 1:300), OCT3/4 (SC-5279, 1:300), SOX-2 (SC-20088, 1:300), IKKa (19930S, 1:1000), IKBa (4814S, 1:1000), p-IKBa (2859S, 1:1000), p65 (D14E12, 1:1000), p-p65 (3033S, 1:1000), BAX (CST2772S, 1:1000), NOTCH1 (SC373891, 1:1000), T-AKT (92112, 1:800), p-AKT (SC-29315, 1:300), BCL-2 (SC-492, 1:300), TACE (SC-6416, 1:300), a-tubulin (SC-23975, 1:300), HES1 (SC-13844, 1:500), b-actin (SC-47778, 1:1000) (Santa Cruz Biotechnology, Dallas, TX, USA), and intracellular domain (ab83232, 1:1000) (Abcam, Cambridge, UK) overnight at 4 °C. A 2 h incubation was followed by rabbit (SC-2004), anti-mouse (SC-2005), -goat (SC-2020), or -rat (SC-2006) IgGs (1:1000) that were tagged with horseradish peroxidase (Santa Cruz Biotechnology). A high-performance chemiluminescence kit (Amersham Bioscience, Piscataway, NJ, USA) was used to track protein signals.
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