Goat anti rabbit immunoglobulin g igg

TOCP (purity >99%) was obtained from BDH Chemicals Company Limited (Poole, UK). Dulbecco modified Eagle medium (DMEM)/F12 (1:1) medium and B27 supplement were purchased from Gibco BRL (Caithersburg, MD, USA). Basic fibroblast growth factor (bFGF) was purchased from R&D Systems, Minneapolis, MN, USA. Bafilomycin A₁ (Baf A₁), N-acetylcysteine (NAC), melatonin, and 4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cell-Light™ 5-ethynyl-2′-deoxyuridine (EdU) Apollo^®488 in vitro Imaging Kit (100T) was purchased from RiboBio Company Limited (Guangzhou, China). Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit was purchased from Abcam, Cambridge, MA, USA. The primary antibodies: rabbit anti–light chain 3 beta (LC3B), rabbit anti–neuronal class III β-tubulin (Tuj-1), and rabbit anti–glial fibrillary acidic protein (GFAP) were purchased from Cell Signaling Technology, Danvers, MA, USA. Monoclonal anti–β-actin, goat anti–rabbit immunoglobulin G (IgG), and anti–mouse IgG were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicinchoninic acid assay protein assay kit was purchased from Pierce Biotechnology Inc., Rockford, IL, USA. The 2′,7′-dichlorodihydro-fluorescein diacetate (H₂DCFDA) and dihydroethidium (DHE) were purchased from Molecular Probes, Eugene, OR, USA.

Radioimmunoprecipitation assay buffer (20 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1% Nonidet P-40 (NP-40), 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin) was added to samples for protein extraction. For Western blotting, 30 µg of the total lysates was separated using 6% to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). After blocking with dried nonfat milk for 1 h, the membrane was incubated overnight with primary antibodies at 1:3000 dilution. The primary antibodies and antibodies against phosphorylated epitopes used in this study were mTOR, phospho-mTOR (Ser2448), p70S6K, and phospho-p70S6K (all purchased from Cell Signaling Technologies, Danvers, MA, USA). β-actin (1:5000 dilution; Sigma Aldrich, St. Louis, MO, USA) was used as the internal control. Horseradish-peroxidase-conjugated goat anti-mouse IgG (Sigma Aldrich, St. Louis, MO, USA) and goat anti-rabbit Immunoglobulin G (IgG) (Sigma Aldrich, St. Louis, MO, USA) were used as secondary antibodies. Western Lightning^® Plus-Enhanced Chemiluminescence (ECL) Substrates (PerkinElmer, Inc., Boston, MA, USA) were used to visualize the proteins.