Enhanced chemi luminescence (ecl)

Cells were lysed in Laemmli buffer (Bio-Rad) with β-mercaptoethanol (Scharlau) and protease inhibitor and phosphatase-stop (Roche). Samples were sonicated on a Diagenode Bioruptor system (10 min), heated (99°C, 5 min) and centrifuged (4°C, 5 min) before loaded (equivalent to 100,000 cells per well) onto precast TGX gels (Bio-Rad). Membrane was blocked with 5% dry milk powder solution (w/v) and then incubated with primary and HRP-conjugated secondary antibodies in 1% blocking solution (w/v). Membrane was developed using ECL (Biological Industries). Resulting blots were imaged on a ChemiDoc XRS+ (Bio-Rad) and bands of selected epigenetic biomarkers were normalised via expression of GAPDH. Fold change was determined by normalisation to expression from day 0 samples.

The total proteins, nuclear proteins or cytoplasmic proteins were extracted from the exosomes or cortex tissues of the infarcted cerebral hemisphere according to the manufacturer's instructions (Beyotime). The amount of protein was quantified by the BCA assay. Equal amounts of protein were electrophoresed by 10% SDS‐PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes. After blocking, membranes were incubated with the following primary antibodies, including CD63 (1:500; Abcam), CD9 (1:500; Abcam), CD81 (1:500; Abcam), TLR4 (1:1000; Affinity Biosciences), NF‐κB p65 (1:1000; Affinity Biosciences), HMGB1 (1:1000; Abcam), GAPDH (1:2000; Affinity Biosciences), MyD88 (1:1000; Affinity Biosciences), β‐actin (1:2000; Proteintech), Histone H3 (1:2000; Affinity Biosciences) and the secondary HRP‐labelled antibodies (1:5000; Proteintech). Band visualization was enhanced with chemiluminescence (ECL; Biological Industries), and the results were processed with a Chemiluminescent Imaging and Analysis System (Biolight Biotechnology Co., Ltd.).

Cell cultures were lysed with RIPA lysis buffer (Tris-HCl 10 mmol/L, EDTA 5 mmol/L, NaCl 50 mmol/L, 1% deoxycholic acid and 1% triton X-100, pH 7.4). Protein concentration was determined by the Bradford method (BioRad protein assay). Equal amounts of proteins from cell extracts were separated by SDS-PAGE 8–15%, electro-transferred to PVDF membranes and blocked with 5% milk. Primary antibodies were used against type I collagen 1:2000 (cat #ABT123 Millipore), p62 1:2000 (cat #5114 Cell Signaling), osteopontin 1:2000 (cat #ab8448 Abcam), BECLin1 1:2000 (cat #3738 Cell Signaling), α-SMA 1:20000 (cat #ab7817 Abcam), GAPDH 1:50000 (cat #8795 Sigma), SM22 1:10000 (cat #ab14106 Abcam), LC3-I/II 1:1000 (cat #2775 Cell Signaling). Membranes were re-blotted with a horseradish peroxidase-linked secondary antibody 1:5000 (mouse cat #402335 and rabbit cat#401315 Merck). Bands were detected using ECL (Biological Industries) and luminescence was assessed using a digital imaging system (Syngene). Quantification of the bands by densitometry was performed using UN-SCAN-IT gel software.

Samples were separated on 12% SDS-PAGE gels. The proteins were then electrotransferred onto Immobilon-P transfer membrane (Millipore, Millipore, Bradford, MA, USA) and blotted with ECLhCZIBPBHhf-iFeN8r/" target="_blank">anti-PARP (Cell Signaling Technology, Inc, Boston, USA; 1 : 1,000). Band visualization was achieved using an enhanced chemiluminescence kit (ECL, Biological Industries).

Cells were lysed in a buffer containing 2% SDS and 67 mM Tris-HCl, pH 6.8, before being sonicated for 20 seconds twice. The same amount (30 μg) of protein (measured by the Lowry method) from each cell lysate were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on to Immobilon-P membranes for 2 hours at 60 V. The membranes were incubated with the following antibodies: anti-p21Waf-1 mouse monoclonal antibody (diluted 1:200; Ab-1, OP64, Calbiochem); anti-UBC9 (UBE2I) rabbit polyclonal antibody (diluted 1:300; ab-30505, Abcam); anti-SENP2 rabbit polyclonal antibody (diluted 1:3,000; made by immunizing a rabbit with the antigen containing the amimoacids 111–358 of human SENP2); anti-SUMO-1 (FL-101) rabbit polyclonal antibody (sc-9060, Santa Cruz Biotechnology); and anti-actin (Clone C4) mouse monoclonal antibody (diluted 1:5,000; 691001, MP Biomedicals). After incubation with the appropriate peroxidase-coupled secondary antibody (diluted 1:2,500; Bio-Rad), immunocomplexes were detected by enhanced chemiluminescence (ECL) (Biological Industries).

Protein samples were prepared by trypsinization of HEK293 cells (1 × 10⁶ cells). The cells were washed by PBS buffer and then lysed by adding 2x Laemmli sample buffer supplemented with 2M urea and boiled for 10 min. Samples of cell extracts were separated by SDS-PAGE (GeBARunner, DNR Bio-Imaging Systems) under reducing conditions using 4–12% gradient polyacrylamide gels (DNR Bio-Imaging Systems) according to manufacturer's instructions.
The samples were then electrophoretically transferred for 16 hr in 4°C using wet transfer standard protocol to the nitrocellulose membranes. Blots were blocked for 1 hr in PBST (PBS buffer and 0.1% Tween) containing 5% skim milk, followed overnight incubation at 4°C with anti-GFP (Cell signaling, RRID:AB_390710) and anti-mCherry (Clontech, RRID:AB_10013483) primary antibodies. Blots were washed three times and incubated for 1 hr at room temperature with the secondary antibody (HRP-conjugated goat anti-rabbit IgG, Jacksonimmuno, RRID:AB_2307391). Immunoreactive bands were visualized by the enhanced chemiluminescence method (ECL) (Biological Industries) according to standard procedures.