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Dernière publication 01/10/2024

Association of DNA methylation and demethylation with the functional outcome of

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...

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    [title] => Association of DNA methylation and demethylation with the functional outcome of 
    [paragraph] => Association of DNA methylation and demethylation with the functional outcome of stroke in a hyperinflammatory state
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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)
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