Phosphorylated akt

Total cellular proteins were extracted with radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Jiangsu, China). The protein concentration was measured using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Briefly, equivalent quantities of protein sample (40 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electrotransferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Subsequently, the membrane was blocked at 4°C for 1 h with TBS containing 0.05% Tween-20 (TBST) buffer with 5% non-fat milk and then incubated with the following primary antibodies against HMGA2 (1:1,000; cat. no. ab97276; Abcam), AKT (1:1,000; cat. no. sc-8312; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphorylated (p)-AKT (1:1,000; cat. no. sc-33437; Santa Cruz Biotechnology, Inc.), mammalian target of rapamycin (1:500, mTOR; cat. no. sc-8319; Santa Cruz Biotechnology, Inc.) and p-mTOR (1:500, cat. no. sc-101738; Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following incubation with secondary antibodies conjugated with horseradish peroxidase (LK2001 and LK2003; 1:100; Sungene Biotech, Co., Ltd., Tianjin, China) for 1 h at room temperature, the bands were examined using an enhanced chemiluminescence system (MultiSciences Biotech Co., Ltd., Hangzhou, China).

Total protein was extracted from the HT22 cells following transfection for 2 days using RIPA lysis buffer, and then the protein concentration was quantified by a BCA kit (both from Beyotime Institute of Biotechnology). Next, amount of protein (50 μg) was loaded onto gels and subjected to SDS-PAGE (10% gel) prior to transfer to a poly-vinylidene difluoride membrane (0.45 μm; EMD Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at 37°C and probed with primary antibodies against B-cell lymphoma 2-associated X protein (Bax; cat. no. sc-6236; 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), phosphoinositide 3-kinase (PI3K, cat. no. sc-7174; 1:2,000; Santa Cruz Biotechnology), phosphorylated (p)-Akt (cat. no. sc-7985-R; 1:500; Santa Cruz Biotechnology), PTEN, nuclear factor (NF)-κB (cat. no. sc-109; 1:500; Santa Cruz Biotechnology) and GAPDH (cat. no. 5174; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. The membranes were then washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074; 1:5,000; Cell Signaling Technology, Inc.) at 37°C for 1 h. Protein bands were visualized using BeyoECL Star (Beyotime Institute of Biotechnology) and quantified by Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

After 48 h following transfection, total protein from cells was isolated using radioimmunoprecipitation assay buffer with protease inhibitor Cocktail (Pierce; Thermo Fisher Scientific, Inc.). The protein concentration was determined using a BCA protein assay kit. Total proteins (20 µg) were separated via 10% SDS-PAGE and then transferred onto to polyvinylidene difluoride membranes (BD Pharmingen; BD Biosciences). The membranes were blocked with 5% non-fat milk at 4°C overnight, and incubated with primary antibodies against PTEN (cat. no. sc-7974, 1:1,000, Santa Cruz Biotechnology, Inc.), phosphorylated (p)-AKT (cat. no. sc-7985-R, 1:1,000, Santa Cruz Biotechnology, Inc.) and AKT (cat. no. sc-5298, 1:1,000, Santa Cruz Biotechnology, Inc.) at 4°C overnight. β-actin (cat. no. A-5441, 1:1,000, Sigma-Aldrich; Merck KGaA) served as an internal control. Horseradish peroxidase-conjugated (cat. no. sc-2031, 1:5,000, Santa Cruz Biotechnology, Inc.) antibodies were used as the secondary antibodies, incubating with the secondary antibody for 1 h at room temperature. The protein bands were scanned on the using the ChemiDocXRS + Imaging System (Bio-Rad Laboratories, Inc.). The band intensity was quantified using Quantity One v4.6.2 software (Bio-Rad Laboratories, Inc.). All the experiments were performed in triplicate.