Association of DNA methylation and demethylation with the functional outcome of stroke in a hyperinflammatory state

Authors


Lab

Journal
Neural Regeneration Research

Abstract
Brain samples collected at 24 hours after tMCAO were lysed using RIPA buffer (RIPA:PMSF = 100:1). Cells were broken up in a grinder and the resultant liquid was then centrifuged. After centrifugation at 13,400 xgat 4C for 30 minutes, the supernatant was collected as a protein extract. The concentrations of proteins were determined using a BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA, Cat# 23225). The supernatant was added into 1x SDS loading buffer (APPLYGEN, Beijing, China, Cat# B1012-5) with a final concentration of 0.1%. The SDS samples were boiled for 10 minutes before loading. Proteins (25 g) were fractionated by electrophoresis on 10% Nupage Bis-Tris gels (APPLYGEN, Cat# T202110) and then transferred onto a polyvinylidene fluoride membrane (Merck Millipore, Billerica, MA, USA, Cat# IPFL00010). Then, membranes were incubated with primary antibodies against GADPH (mouse, 1:2000, ZSGB-BIO, Beijing, China, Cat# TA-08, RRID: AB_2747414), DNMT3A (mouse, 1:1000, Abcam, Cambridge, UK, Cat# ab2850, RRID: AB_303355), DNMT3B (mouse, 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA, Cat# sc-81252, RRID: AB_1122311), DNMT1 (mouse, 1:500, Santa Cruz Biotechnology, Cat# sc-271729, RRID: AB_10710384), TET1 (mouse, 1:500, Santa Cruz Biotechnology, Cat# sc-293186), TET2 (rabbit, 1:1000, Cell Signaling Technology, Danvers, MA, USA, Cat# 36449, RRID: AB_2799102), IL-1Beta (mouse, 1:500, Cell Signaling Technology, Cat# 12242, RRID: AB_2715503), IL-6 (mouse, 1:500, Santa Cruz Biotechnology, Cat# sc-28343, RRID: AB_627805), NF-kBeta (mouse, 1:500, Santa Cruz Biotechnology, Cat# sc-8008, RRID: AB_628017) and p-NF-kBeta (mouse, 1:500, Santa Cruz Biotechnology, Cat# sc-136548, RRID: AB_10610391) at 4C overnight. After being washed three times with Tris-buffered saline with Tween 20, the membranes were incubated for 1-2 hours at room temperature with horseradish peroxidase (HRP)-conjugated donkey anti-mouse IgG (1:5000, Jackson Immunoresearch Laboratories, West Grove, PA, USA, Cat# 711-035-151, RRID: AB 2340771) or HRP-conjugated donkey anti-rabbit IgG (1:5000, Jackson Immunoresearch Laboratories, Cat# 711-035-152, RRID: AB 10015282). Protein levels were determined using enhanced chemiluminescence HRP detection reagents (Solarbio, Cat# PE0010) and Image Reader (Syngene, Cambridge, UK, G:BOX Chemi XX9).

Keywords/Topics
: DNA demethylation ; DNA methylation ; DNMT3A ; functional outcome ; hyperinflammatory state ; interleukin ; neuroinflammation ; stroke

BIOSEB Instruments Used:
Automatic Foot misplacement apparatus (BIO-FMA)

Source :

https://journals.lww.com/nrronline/fulltext/2024/10000/association_of_dna_methylation_demethylation_with.24.aspx

DEMANDE DE PUBLICATION

Merci de votre intérêt pour notre gamme de produits et de votre demande pour cette publication qui vous sera envoyée si l'équipe de recherche et la revue le permettent. Notre équipe commerciale vous contactera dans les plus brefs délais.