Anti app

Gallic acid, quercetin, dimethyl sulfoxide (DMSO), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12 medium, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Geneticin (G418) was purchased from Invitrogen (San Diego, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) was purchased from Bio Basic (Markham, Ontario, Canada). Trizol reagent was purchased from Invitrogen (Carlsbad, CA, USA). Phosphate buffer saline (PBS) was HyClone^TM and purchased from GE Healthcare Bio-Sciences, USA. EmbryoMax^® non-essential amino acids (NEAA) solution was purchased from Millipore^® (Burlington, MA, USA). Penicillin/Streptomycin solution was purchased from Gibco (Waltham, MA, USA). Cell lysis buffer (10X), anti-APP, anti-β-actin, and horseradish peroxidase-coupled secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Methanol was purchased from Merck (Darmstadt, Germany).

Aβ_25–35 (A4559) and d-galactose (G0625) were purchased from Sigma-Aldrich (USA). Aricept was purchased from Eisai Pharmaceutical Co., Ltd. (China). The detection kits for superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), Na⁺/K⁺-ATPase, acetylcholine (ACh), acetylcholinesterase (AChE), nitric oxide (NO), and glutamate (Glu) were all purchased from Nanjing Jiancheng Bioengineering Institute (China). The level of γ-aminobutyric acid (GABA) was measured using a kit purchased from Bogoo Biotechnology Co., Ltd. The following primary antibodies were used for the present study: anti-APP (Cell Signaling, Cat. #2452), anti-SYP (Cell Signaling, Cat. #5461), anti-caspase-3 (Cell Signaling, Cat. #9665), anti-GAPDH (Cell Signaling, Cat. #5174), and anti-p-tau ser404 (Epitomics, Cat. #2691-1).

The expression levels of p-AMPK, p-P65NF-κB, p-P38MAPK, Bace1, and APP were analyzed by Western blotting. The protein samples were heated at 100 °C for 5 min with a loading buffer containing 0.125 M Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.002% bromophenol blue. The samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene (PVDF) membranes. The membranes were incubated with 3% bovine serum albumin (BSA) in Tris-buffered saline with Tween (TBST) (10 mmol/L Tris at pH 7.5, 150 mmol/L NaCl, 0.05% Tween-20) and probed with corresponding primary antibodies (anti-p-AMPK, anti-p-P38MAPK, anti-p-P65NF-κB, anti-BACE1, and anti-APP; Cell Signaling Technology, Boston, USA) at 4 °C overnight. After incubation with horseradish peroxidase-coupled secondary antibodies for 2 h at room temperature, the protein bands were quantified by densitometry (Syngene, UK).

The samples of the cerebral cortex of rats in four groups were homogenized and lysed by RIPA containing 1% protease inhibitor cocktail and 1% PMSF (Beyotime, Shanghai, China). Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis and transferred onto PVDF membrane blocked with 5% skimmed milk as previously described (Li et al., 2018 (link)). A pre-stained protein marker (Thermo Fisher Scientific, MA, United States) was run in parallel to detect the molecular weight of proteins. Proteins were probed with appropriate antibodies including anti-TLR4 (1:1000, abs132000; Absin Bioscience Inc., Shanghai, China), anti-CD14 (1:500, abs121538; Absin Bioscience Inc., Shanghai, China), anti-IRAK1 (1:1000, abs143411; Absin Bioscience Inc., Shanghai, China), anti-p65 (1:1000, no. 8242; Cell Signaling Technology, United States), anti-pp65 (1:1000, no.3033; Cell Signaling Technology, United States) anti-BACE1 (1:1000, no. 5606; Cell Signaling Technology, United States), anti-APP (1:1000, no. 2452; Cell Signaling Technology, United States) and anti-GAPDH (1:1000, AB-P-R001, Goodhere Biotechnology Co., Hangzhou, China). The data were quantified using the Image Studio Lite ver. 5.2 software.

The following reagents and materials were used. From Cell Signaling technology (Danvers, MA, USA): anti-APP (ref. 2450), anti-FAK (ref. 3285), anti-phospho-FAK (Y576/577) (ref. 3281), anti-Akt (ref. 4685), anti-phospho-Akt (S473) (ref. 4060), anti-PDK1 (ref. 3062), anti-phospho-PDK1 (S241) (ref. 3061), anti-phospho-PKC pan (T514) (ref. 9379), anti-p38 MAPK (ref. 9212), anti-phospho-p38 MAPK (T180/Y182) (ref. 9211), anti-MEK1/2 (ref. 9126), anti-phospho-MEK1/2 (S217/221) (ref. 9154), anti-ERK1/2 (ref. 9102), anti-phospho-ERK1/2 (T202/y204) (ref. 4370), anti-CREB (ref. 9104), anti-IκBα (ref. 4814S), anti-phospho-IκBα (S32) (ref. 2859), anti-NFκB p65 (ref. 8242), anti-phospho-NFκB p65 (ref. 3033S), anti-SEK1 (ref. 9152), and anti-phospho-SEK1 (S257/T261) (Ref. 9156). Anti-PKC-pan was from SigmaAldrich (St Louis, MO, USA) (ref. SAB4502356). Electrophoresis reagents were purchased from Bio-Rad (Hercules, CA, USA) and trypsin from Promega (Madison, WI, USA).

Western blot was performed as described before (Sun et al., 2015 (link)). Briefly, the PVDF membranes were incubated with anti-Cofilin-2 (Santa Cruz, sc-166985), anti-Cathepsin B (Cell Signal Technology, 31718), anti-Triosephosphate isomerase (Abcam, ab28760), anti-Clusterin (CST, 34642), anti-ITI-H4 (Santa Cruz, sc-515353), anti-APP (Cell Signal Technology, 29765), Anti-sAPPα (IBL, 11088), Anti-sAPPβ (IBL, 18957) Anti-BACE1 (Abcam, ab2077), anti-ADAM10 (Cell Signal Technology, 14194) and anti-β-actin (Cell Signal Technology, 3700) overnight at 4°C. After washed with TBST, HRP-conjugated secondary antibodies (1:10,000) were applied at room temperature for 1 h. The signals were detected by a ChemiDoc MP system (Bio-Rad) and analyzed by ImageJ software.