Dykddddk tag

Protein was extracted from GSC cells or GBM cells. Primary antibodies to the following were used: GAPDH (Cell Signaling Technology, 5174), β-actin (Cell Signaling Technology, 14074), N-cadherin (Cell Signaling Technology, 13116), E-cadherin (Proteintech, 20874-1-AP), CD44 (Proteintech, 15675-1-AP), AKT (Cell Signaling Technology, 9272), phospho-Akt (Ser473, Cell Signaling Technology, 4060), CDK1/cdc2 (Cell Signaling Technology, 9116), phospho-CDK1/cdc2 (Tyr15) (Cell Signaling Technology, 4539), cyclin B1 (Cell Signaling Technology, 12231), P21 (Cell Signaling Technology, 2947), IGF2BP3 (Abcam, ab177477), ubiquitin (Cell Signaling Technology, 3936), DYKDDDDK Tag (Cell Signaling Technology, 14793), His-tag (Cell Signaling Technology, 12698), CUL2 (Proteintech, 10981-2-AP; Santa, sc-166506), and RPN2 (Abcam, ab244399).

Cells were harvested in RIPA lysis buffer, and protein was quantified with the BCA assay (Beyotime). Whole-cell lysates containing 15 μg total protein were loaded onto 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gels, separated by electrophoresis, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Nonspecific binding on the membranes was blocked with 5% bovine serum albumin (BSA) in TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Membranes were then probed with primary antibodies (1 : 1000) against mouse FoxO1, DYKDDDDK Tag (Cell Signaling Technology, Danvers, MA, USA) and α-tubulin (Sigma) in blocking solution overnight at 4 °C. After washing with TBS-T, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. Protein bands were visualized by exposing to an enhanced chemiluminescence detection system (LAS-4000 imager, Fujifilm, Tokyo, Japan) using the SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA). Densitometry analyses were performed using ImageJ 1.42q software (National Institutes of Health), and the values for target proteins were normalized to α-tubulin as the endogenous control.

Malondialdehyde detection by lipid peroxidation MDA assay kit and ROS detection by reactive oxygen species assay Kit were provided by Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Polyvinylidene fluoride (PVDF) was from Millipore (Billerica, MA, USA). Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were products from Gibco (Australia). Lipofectamine™ 3000 was from Invitrogen (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The primary antibodies for cleaved caspase-9, cleaved caspase-3, phospho-p44/42 MAPK (p-ERK1/2), AKT, Phospho-Akt (Thr308), Phospho-Akt (Ser473), DYKDDDDK Tag, snail and β-actin were purchased from Cell Signaling Technology (Danvers, MA, America). The primary antibodies for Cyt c and Cyt b₅ were purchased from Abcom (Cambridge Biomedical Campus, Cambridge, UK). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Aladdin (Shanghai, China). The phosphatase inhibitor cocktail and protein inhibitor cocktail were from Beijing ComWin Biotech Co., Ltd. (Beijing, China). Confocal laser imaging was recorded on the Zeiss LSM 880, Western blot analysis was performed on the Tanon 5200, and OD value measurement was detected using SpectraMax Absorbance Reader.