Anti beclin 1

High‐glucose (25 mM) Dulbecco's modified Eagle's (DMEM‐H), foetal bovine serum (FBS), antibiotics (penicillin and streptomycin), and Lipofectamine2000 were obtained from Thermo Fisher Scientific. Methyl viologen dichloride hydrate (paraquat, PQ), dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA), DAPI, acridine orange (AO), dansylcadaverine (MDC), and NAO dye were purchased from Sigma (MO, USA). Rapamycin (RAPA) and 3‐methyladenine (3‐MA) were purchased from Selleckchem. ST2825 was purchased from MedChemExpress. All molecular biological reagents were obtained from New England Biolabs. Primary antibodies against mTOR, phosphor(P)‐mTOR, P‐p53, Drp1, Tom20, cleaved‐caspase 3, and Bcl‐2 were purchased from Cell Signalling Technology. Anti‐Ki67, phosphorylated histone H2A.X (P‐γH2A.X), CD3, and CD68, and Alexa Fluor® 488‐conjugated or Alexa Fluor® 594‐conjugated second antibody were purchased from Abcam. Anti‐LC3B, SQSTM1/P62, and MyD88 were purchased from Sigma. Anti‐Beclin 1, CDK2, GPX1, 8‐OHdG, OPA1, and Fis1 were purchased from Bioss (Woburn, MA, USA). Anti‐SOD1, p53, PARP1, NDUFB8, and UQCRFS1 were purchased from Proteintech. Secondary antibodies horseradish peroxidase‐conjugated goat anti‐rabbit or mouse IgG was purchased from ZSGB‐Bio.

Equal amounts of protein (30 µg) extracted from the cells or brain tissues were separated using 8%-15%SDS-PAGE gels and transferred to PVDF membranes (Millipore; Germany). Then, the membranes were blocked with 5% BSA for 1h and incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibody for 2h at room temperature. Primary antibodies against the following proteins were from Cell Signaling Technology, (Beverly, MA, USA): anti-SQSTM1/p62 (no.8025T), anti-Phospho-mTOR (no.5536T), anti-TFEB (no.37785S), anti-mTOR (no.2983S). Anti-LC3B (no. A9A503500321), anti-p70S6K (no. A9A373640725), anti-Phospho-p70S6K (no. A9A303700725), anti-β-actin (no.61A00848) were purchased from MultiSciences (Zhejiang Province, China). Anti-Beclin1 (no. AH11208656) and anti-ATG5/APG5L (no. AB04011456,) were purchased from Bioss (Woburn, MA, USA). Anti-GAPDH (no.LS191936) and anti-Cathepsin D (no.050818190315) were purchased from Servicebio (Wuhan, China) and Beyotime Biotechnology (Jiangsu, China), respectively. The membranes were visualized using an ECL kit (4A Biotech, Beijing, China) and a chemiluminescence instrument. The integrated optical density (IOD) was measured using the Image Pro Plus software (Version 6.0; Media Cybernetics), and GAPDH, β-actin or LaminA/C was used to normalize protein loading.