Pparγ transcription factor assay kit

Nuclear extracts were prepared from OPCs using NE-PE Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher, 78833, Pittsburgh, PA, USA). PPAR activity was assessed using the PPAR (α, δ, γ) Transcription Factor Assay Kit (Abcam, ab133113, Toronto, ON, Canada) or the PPARγ Transcription Factor Assay Kit (Cayman Chemical, 10006855, Ann Arbor, MI, USA) following the manufacturers’ instructions.

PPARγ DNA binding activity was measured using a PPARγ transcription factor assay kit (Cayman Chemical, USA). Briefly, 10 μg of extracted nuclear proteins were added to wells containing immobilized dsDNA sequences corresponding to the peroxisome proliferator response element. Bound PPARγ was detected by the addition of specific antibodies against PPARγ. Relative PPARγ DNA binding activity was determined by normalizing the measurements obtained for cells transfected with Yulink-shRNA to those obtained for cells treated with Ctrl vector. Rosiglitazone (Santa Cruz), pioglitazone (Sigma), GW7647 (Sigma), and GW0742 (Sigma) were stored in the dark at − 20 °C. Working solutions were prepared by diluting the stock solution in media. Stable cells transfected with Yulink-shRNA or Ctrl vector were treated with or without agonist for 6 h, 12 h, or 2 days. RNA was subsequently isolated from cells using Quick-RNA MiniPrep (Zymo Research, USA), according to the manufacturer’s instructions.

Curcumin, GW9662, Aβ_1–42, and Griess reagent were purchased from Sigma. Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), and Opti- Minimum Essential Medium (MEM) were producted by Gibco. PPARγ siRNA was synthesized by Invitrogen. Lipofectamine LTX and Plus Reagent was produced by Invitrogen. Choline acetyltransferase (ChAT), glial fibrillary acidic protein (GFAP), Iba-1, NF-κB p65, IκBα, and PPARγ antibodies were obtained from Abcam. IL-1β, TNF-α, and COX-2 ELISA kits were purchased from R&D Company. A choline/acetylcholine (Ach) assay kit was supplied by Abcam. A ChAT ELISA kit was obtained from MyBioSource Inc. A PPARγ transcription factor assay kit and PPARγ ligand binding domain (human recombinant) were purchased from Cayman Chemical. A co-immunoprecipitation (Co-IP) kit produced by Pierce was used, and LDH assay kit was supplied by Nanjing Jiancheng Bioengineering Institute.

Nuclear protein was obtained by using NE-PER nuclear and cytoplasmic extraction kit (Pierce, Austin, TX). PPAR-γ activation was detecting using PPAR-γ transcription factor assay kit (Cayman, Ann Arbor, MI).

Ten wide-type mice from the vehicle and 0.7 g/kg/d ethanol groups were euthanized and exsanguinated at the end of 8 weeks of feeding. The brains were removed and cut into six 1.75 mm-thick coronal sections. Under the microscope, the cortical tissues were collected from the parietal and temporal lobes. The nuclear fraction from the cortical tissues was isolated using FOCUS SubCell Kit (G-Biosciences, St. Louis, MO, USA) following the manufacturer’s instructions. The protein concentration was measured using a BCA assay (Thermo Scientific, Plainville, MA, USA). Nuclear PPARγ protein expression was measured by Western blot analysis as described below. Nuclear PPARγ DNA-binding activity was determined by a PPARγ transcription factor assay kit (Cayman, Ann Arbor, MI, USA) following the manufacturer’s protocol. In brief, 120 µg nuclear proteins were incubated with a biotin-labeled DNA probe containing a PPAR-specific double-stranded consensus sequence and a single-stranded capture region. The samples were digested with a double-stranded DNA-specific nuclease and subsequently transferred to a 96-well plate coated with single-stranded DNA complementary to the capture region. A chemiluminescent alkaline phosphatase substrate was added, and the output signal was measured using FLUOstar Omega microplate reader (BMG LABTECH, Cary, NC, USA).