Gtx213111 01

The following chemicals and reagents were procured from Sigma-Aldrich (St. Louis, MO, USA): AITC, methylene blue, propidium iodide (PI), ethanol, DMSO, and isopropanol. A concentration of 10 mM AITC was prepared by dissolving it in DMSO and storing it at 4 °C. RPMI 1640 and fetal bovine serum (FBS) were obtained from Biological Industries (Beit-Haemek, Israel), while RIPA buffer was purchased from Millipore (Burlington, MA, USA). The present study utilized primary antibodies against KDM8 (AVIVA SYSTEMS BIOLOGY; ARP58120_P050), H3K36me2 (GeneTex; GTX54108), Cyclin A1 (GeneTex; GTX02524), and GAPDH (GeneTex; GTX100118), as well as secondary antibodies (GeneTex; GTX213110-01, GTX213111-01).

Mice and rats were killed by an overdose of urethane 24 h after dMCAO or craniectomy and then perfused with cold saline to remove circulating albumin from the blood. Isolated brains were coronally sectioned at 2 mm thickness. Motor cortex tissue from the middle two coronal sections (approximately +2 to +6 mm from λ), where infarct occurred or would have occurred, was isolated for Western blotting as described previously (Liao et al., 2019 (link)). In brief, these samples were homogenized in lysis buffer (20 mm Tris, 150 mm NaCl, 1 mm EDTA, and 1% NP-40 adjusted to pH 7.4) containing protease inhibitor (catalog #04693132001, Roche) and centrifuged at 12,000 rpm for 30 min. Proteins (∼30 μg) from the supernatants were heated for 5 min at 100°C, separated by electrophoresis, and transferred to PVDF membranes. After blocking in milk (5% dry milk powder in TBST) for 2 h at room temperature, each membrane was incubated with primary antibodies against α-tubulin (1:5000 in 2% BSA in TBST; catalog #GTX628802, Genetex) and albumin (1:5000 in 2% BSA in TBST; catalog #ab106582, Abcam) overnight at 4°C, washed with TBST, and incubated with goat anti-mouse IgG (1:1000 in 2% BSA in TBST; catalog #GTX213111-01, Genetex) and goat anti-chicken IgY (1:1000 in 2% BSA in TBST; catalog #ab97135, Abcam) secondary antibodies. Western blot images were taken using a ChemiDoc XRS+ System (BIO-RAD).

One hundred μl of 10-fold PBS-diluted normal and HD plasma were coated in a 96-well ELISA plate and incubated at 4 °C overnight. After removing the plasma, the plate was washed by TBST and blocked with 5% BSA in TBS at room temperature for 2 hr. The plate was then probed by the primary SERF#1 antibody (1:100) at 4 °C overnight followed by the secondary HRP-conjugated anti-mouse antibody (1:5,000; GTX213111-01, GeneTex) at room temperature for 1 hr. The signal was finally developed by TMB microwell peroxidase substrate and the reaction was stopped by adding 250 mM HCl. The absorbance at 450 nm was recorded by using SpectraMax M5 microplate reader (Molecular Devices) with SoftMax Pro 5.4. The data was analyzed by GraphPad Prism9.

Western blot assay was performed as described previously³⁹ with the specific antibodies listed below: Anti‐Asialoglycoprotein Receptor 1 antibody (ASGR1‐Ab, ab254262, Abcam Inc., Cambridge, MA), Phospho‐Stat3 (Tyr705)(D3A7) XP Rabbit mab (pSTAT3 Ab, #9145, Cell Signaling Inc., Danvers, MA), Anti‐h/m/rSTAT3 purified mouse monoclonal IgG (STAT3 Ab, #MAB1799, R&D Systems Inc., Minneapolis, MN), GAPDH antibody (GTX100118, GeneTex Inc., CA), Mouse IgG antibody (HRP) (GTX213111‐01, GeneTex Inc., CA), Rabbit IgG antibody (HRP) (GTX213110‐01, GeneTex Inc., CA).

The brain samples were lysed in RIPA lysis buffer with protease inhibitor cocktail (B14001, Bimake) by a tissue homogenizer, followed by ultrasonication and centrifugation. The bicinchoninic acid method was used to determine the concentrations of proteins that were mixed with 5 × Laemmli sample buffer and denatured for 5 min at 95 °C. A total of 30 µg of each sample was separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. The membranes were incubated with blocking buffer (TBST buffer containing 5% skim milk powder) for 60 min at room temperature. Next, the membranes were incubated overnight at 4 °C with the primary antibodies. The primary antibodies were as follows: anti-FTO (1:1000, ab92821, Abcam), anti-ADRB2 (1:1000, ab182136, Abcam), anti-SIRT1 (1:1000, 8469 S, Cell Signaling Technology), anti-c-MYC (9402 S, 1:1000, Cell Signaling Technology), and anti-GAPDH (1:10000, GTX100118, GeneTex), HRP-conjugated secondary anti-rabbit (1:5000, GTX213110-01, GeneTex), and HRP-conjugated secondary anti-mouse (1:5000, GTX213111-01, GeneTex). After incubation for 1 h with the corresponding secondary antibodies, the protein bands were detected by chemiluminescence using an ECL reagent.

The incubated samples were centrifuged at 15,000 rpm for 10 min to separate the soluble proteins in supernatant and the insoluble fibrils in pellet. Each fraction was subjected to a 13% Tris/tricine separating gel with 10% spacing gel and 4% stacking gel for SDS-PAGE and transferred to PVDF membrane (GE) which was then probed with the primary antibody SERF#1 (1:100) and the secondary HRP-conjugated anti-mouse antibody (1:5,000; GTX213111-01, GeneTex). The membrane was then developed with ECL reagent (Millipore) and the signals were detected by ImageQuant LAS 4000.

The incubated samples were centrifuged at 15,000 rpm for 10 min to separate the soluble proteins in supernatant and the insoluble fibrils in pellet. Each fraction was subjected to a 13% Tris/tricine separating gel with 10% spacing gel and 4% stacking gel for SDS-PAGE and transferred to PVDF membrane (GE) which was then probed with the primary antibody SERF#1 (1:100) and the secondary HRP-conjugated anti-mouse antibody (1:5,000; GTX213111-01, GeneTex). The membrane was then developed with ECL reagent (Millipore) and the signals were detected by ImageQuant LAS 4000.