P mtor

EGCG, anti-LC3B antibody, and anti-NeuN antibody were purchased from Sigma-Aldrich (USA). Donkey anti-mouse IgG secondary antibody was obtained from Life Technologies (Thermo Fisher Scientific, United States). Phosphor-AKT (p-AKT), AKT, p-AMPK, AMPK, p-mTOR, mTOR, beclin1, β-actin, and GAPDH were obtained from ABclonal Technology Co., Ltd. (China). P62/SQSTM1 polyclonal antibody was obtained from Proteintech (China). Horseradish peroxidase (HRP)-conjugated secondary antibody was obtained from Cell Signaling Technology (United States). GSK690693, rapamycin, and LY294002 were obtained from MedChemExpress (United States). Fetal bovine serum (FBS) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Gibco (Thermo Fisher Scientific, United States). The antifade mounting medium was obtained from Solarbio (China). RIPA lysis buffer was obtained from Beyotime (China). Protease and phosphatase inhibitor cocktails and NcmBlot blocking buffer were obtained from New Cell and Molecular Biotech (China). BCA protein assay kit was obtained from CWBIO (China). Cell Counting Kit-8 (CCK8) and ECL chemiluminescent HRP substrate A&B were obtained from Antgene (China). All other reagents, unless stated otherwise, were obtained from Biosharp (China).

Cells and clinical tissues were lysed for 30–45 mins on ice in lysis buffer containing freshly added protease inhibitor cocktail (Roche Diagnostics, USA). After BCA quantification (Pierce, USA), the protein was added to 5× loading buffer and boiled at 95°C for 10 min. Equal amounts of protein were separated by SDS-PAGE and blotted onto activated polyvinylidene difluoride membranes (Millipore, USA). After blocking, the membranes were incubated with primary antibodies overnight at 4°C. The antibodies used were as follows: BMX (1:2000, 610793, BD), phospho-Etk (Tyr40) (1:500, #3211, Cell Signaling Technology), AKT1 (1:500, sc-5298, Santa Cruz Biotechnology), p-AKT (1:1000, #4060, Cell Signaling Technology), mTOR (1:1000, A2445, ABclonal), p-mTOR (1:1000, AP0094, ABclonal), STAT3 (1:1000, #9132, Cell Signaling Technology), phospho-STAT3 (Tyr705) (1:1000, #4113, Cell Signaling Technology), β-actin and GAPDH (1:1000, Santa Cruz Biotechnology). Blots were incubated with secondary antibodies coupled to horseradish peroxidase (Thermo Fisher Scientific, USA) for 1 h and visualized using ECL detection (Millipore) on X-ray film. Relative quantitation was analyzed with Quantity One software.

Total cellular proteins from each group were extracted using RIPA lysis buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail. Then, equal amounts (20 μg) of protein determined by a BCA protein assay kit (Thermo Fisher Scientific, USA) were separated using 6%-10% SDS–PAGE gels. The proteins were then transferred to PVDF membranes (0.45 mm, Solarbio, China) at 300 mA for 2 h. And the blots were cut prior to binding with antibodies during blotting. The membranes were blocked with 5% non-fat milk in TBST for 1 h at room temperature and then incubated overnight with primary antibodies at 4 °C. The following antibodies were tested: MUC3A polyclonal antibody (#PA5-95,355, 1:1000, Thermo Fisher Scientific, MA, USA), p-PI3K (#4228), PI3K (#4257), p-Akt (#4060), Akt (#468), p-mTOR (#5536), mTOR (#2983), p53 (#2527) rabbit monoclonal antibodies (1:1000, CST, MA, USA), and p21 (#A19094) and β-tubulin (#A17913) rabbit monoclonal antibodies (1:1000, Abclonal, Wuhan, China). The secondary antibodies were anti-mouse or anti-rabbit antibodies and were conjugated to horseradish peroxidase (HRP) (1:4000, Abclonal, Wuhan, China). The antibodies were used at a 1:4000 dilution and incubated at room temperature for approximately 1 h. The bands were visualized with ECL reagents (Thermo Fisher Scientific, MA, USA) and developed by Omega Lum G (Aplegen, USA).