Anti chat

Herbal materials were purchased from Lee Hoong Kee Limited (Hong Kong) and Eu Yan Sang (Hong Kong). Antibodies including anti-neurofilament-L, anti-neurofilament-M, anti-neurofilament-H, anti-GFAP, anti-βIII-tubulin, anti-β-actin, anti-ChAT, anti-phospho-ERK, anti-ERK, anti-phospho-CREB, and anti-CREB were obtained from Cell Signaling Technology (MA, USA). Chemical markers for high-performance liquid chromatography (HPLC) reference were purchased from Cheungdu Biopurify Phytochemicals Ltd. (China), Fujifilm Wako Chemical Corporation (Japan), and Aobious Inc. (MA, USA). ReNcell culture materials including ReNcell^™ VM neural progenitor cell line (Cat# SCC008) and ReNcell NSC maintenance medium (Cat#SCM005) were purchased from EMD Millipore. Other cell culture materials were purchased from Thermo Fisher Scientific (OH, USA) and Millipore Sigma (MA, USA). SensoLyte^®Thioflavin T Aβ42 Aggregation kit (Cat# AS-72214) were purchased from AnaSpec (CA, USA). COX-1 and COX-2 inhibitor screening kits were from BioVision Inc. (CA, USA).

Antibodies used were as follows: anti-synaptophysin, anti-ChAT, Anti-PSD95, anti-NeuN (Cell Signaling Technology, Beverly, MA); anti-GPX4 (Santa Cruz Biotechnology, Dallas, TX); anti-4-HNE antibody (R&D Systems, Minneapolis, MN); and anti-β-actin antibody (Abcam, Cambridge, MA).
Tissues were homogenized in RIPA buffer (20 mM Tris, pH 7.4, 0.25 M NaCl, 1 mM EDTA, 0.5% NP-40, and 50 mM sodium fluoride) supplemented with protease inhibitors (539,134, EMD Biosciences Inc., San Diego, CA). Levels of specific proteins in tissues were determined by western blots as previously described^{44 (link)}. In brief, 30 µg total protein per sample was separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% BSA then incubated with primary antibody overnight at 4 °C. After incubation with fluorophore-conjugated secondary antibodies (ThermoFisher, MA) for 1 h, bands were detected using an Odyssey scanner (LI-COR, Lincoln, NE). Alternatively, after incubation with primary antibodies, the membranes were incubated with a HRP-conjugated secondary antibody. The bands were visualized using the ECL Kit (RPN2132, GE Healthcare, Piscataway, NJ). The bands were quantified using NIH ImageJ software (ImageJ 1.52a; http://imagej.nih.gov/ij) and normalized to the loading control (β-Actin). The experiments were repeated at least once.