P akt clone d9e

Total protein from SNU478 cells was extracted by PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea), and protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich). Approximately 1.5 μg of proteins were loaded on a capillary-based Western blot system (ProteinSimple, San Jose, CA, USA) and automatically measured each protein level. Finally, each Western band images were acquired and intensities of the target protein with specific molecular size were quantified by Compass for Simple Western v. 6.1.0. (ProteinSimple). Monoclonal antibodies against B-cell lymphoma 2 (Bcl-2; clone 100; BioLegend), phosphorylated protein kinase B (p-Akt; clone D9E; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:100), Akt (clone 11E7; Cell Signaling Technology, Inc.; 1:100), Sex determining region Y-box 2 (Sox-2; clone D6D9; Cell Signaling Technology, Inc.; 1:100), nuclear factor erythroid-2 related factor 2 (Nrf2; clone D1Z9C; Cell Signaling Technology, Inc.; 1:100), Kelch-like ECH-associated protein 1 (Keap1; clone D6B12; Cell Signaling Technology, Inc.; 1:100), and polyclonal antibodies against β-actin (Cell Signaling Technology, Inc.; 1:200) were used for the analysis.