Peroxidase affinipure goat anti rabbit igg

Griess reagent, 5,5’-Dithiobis (2-nitrobenzoic acid) and Lucigenin were purchased from Sigma-Aldrich. Hydrogen peroxide 33% was procured from PanReac AppliChem. The following antibodies were used in this work: Anti-fibronectin was obtained from GeneTex. Anti-MMP-9 and anti-Bcl2 were obtained from Proteintech. Anti-vimentin, anti-MMP-3, Anti-Akt, Anti-p38, Anti-Cleaved PARP1, Anti-p53 (acetyl K382), Anti-JNK, Anti-alpha smooth muscle Actin, Anti-NF-kB p65 (phospho S536), and anti-β catenin were all obtained from Abcam (Cambridge, UK). Anti-cleaved caspase-3, anti-cleaved aspase-9, and anti-cleaved caspase-8 were obtained from Cell Signaling. Rabbit Polyclonal JNK1/2/3 was obtained from ORIGENE. Anti-beta Actin was obtained from Thermo Fisher Scientific. Peroxidase AffiniPure Goat Anti-Rabbit IgG and Peroxidase AffiniPure Goat Anti-Mouse IgG were obtained from Jackson ImmunoResearch Inc. The following ELISA kits were used in this work: nitrotyrosine ELISA kit (Abcam, ab113848), Hydrogen Peroxide Colorimetric/Fluorometric assay kit (Biovision, K265-200), malondialdehyde assay kits (Northwest Life Sciences Specialties, NWK-MDA01) and Superoxide Dismutase assay kit (Cayman chemical, 706002).

Collected exosomes were lysed and incubated on ice for 30 min. Thereafter, they were vortexed every 10 min to fragment the samples. Following centrifugation at 10000–14000 g for 10 min at 4°C, the supernatant was collected and stored at −80°C. The absorbance of the sample was read at 562 nm using an enzyme-labeled meter (TECAN, Infinite M100 PRO). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to polyvinylidene fluoride (PVDF) (Millipore, MA, United States, #IPVH00010) membranes. The membranes were probed overnight with the primary antibody (CD63, rabbit polyclonal antibody; 26 kDa; 1:1000 dilution with 5% non-fat milk; Proteintech Group, Chicago, IL, United States; #25682-1-Ig) at 4°C. Thereafter, the membranes were probed with the secondary antibody (Peroxidase AffiniPure Goat Anti-Rabbit IgG; 1:10000 dilution; Jackson; #111-035-045) for 2 h at 37°C. Chemiluminescence corresponding to proteins was visualized using the Millipore ECL system (Tanon 4600, Shanghai, China).

To measure the expression of VEGF-A and VEGFR2, 3-h and 7-day ethanol-treated MBMVECs, cerebral cortex, and the peri-infarct cortex were homogenized in ice-cold lysis buffer (10 mM EDTA, 0.1% Tween-20, 1%Triton, 0.1% mercaptoethanol, 150 mM NaCl, 50 mM Tris HCl, 5 μg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 5 μg/ml aprotinin, pH 7.4). Homogenates were centrifuged at 12,000 RPM for 20 min at 4°C. The protein concentration of the supernatants was measured with the Bradford method (Bio-Rad, CA, USA). SDS polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 10% gel. Following SDS-PAGE, the proteins were transferred onto the polyvinylidene difluoride membrane. Immunoblotting was performed using rabbit anti-VEGF-A (ab46154; Abcam), rabbit anti-VEGFR2 (2479s; Cell Signaling), and mouse anti-GAPDH (sc-32233; Santa Cruz) as primary antibodies and Peroxidase-AffiniPure Goat Anti-Rabbit IgG (111-035-144; Jackson) and anti-mouse IgG HRP-linked antibody (7076S; Cell Signaling) as the secondary antibodies. The target proteins were subsequently detected with the enhanced chemiluminescence (ECL) kit (Pierce Chemical, IL). The band densities were analyzed using ChemiDoc™ MP Imaging System (Bio-Rad). For quantification, protein expression of VEGF-A and VEGFR2 was normalized to GAPDH and expressed as percentage changes to the control.

Proteins from control or treated podocytes were extracted using ice-cold 1X RIPA buffer (Abcam, Cambridge, UK) enriched with protease and phosphatase inhibitors (Sigma Aldrich, St. Louis, MO, USA). Immunoblotting was performed on aliquots containing 15 μg/lane of proteins resolved on 8–15% SDS-PAGE as previously described^{16 (link)}. Protein expression was quantified as the ratio of a specific band to β-actin. The values are represented as the fold change compared to the control group. The primary antibodies used were as follows: anti-claudin-1 (1:2000, #51-9000, Invitrogen), anti-SIRT1 (1:2000, #9475, Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-β-actin (1:10,000, ab6276, Abcam, Cambridge, UK). The following secondary antibodies were used: peroxidase AffiniPure goat anti-rabbit IgG (1:10,000, 111-035-003, Jackson ImmunoResearch Laboratories, Baltimore, MD, USA) and peroxidase AffiniPure goat anti-mouse IgG (1:10,000, 115-035-003, Jackson ImmunoResearch Laboratories).

Protein samples were prepared with reducing Laemmli buffer and boiled for 10 min at 95°C, and then separated on NuPAGE 4–12% Bis-Tris Mini protein gel (Thermo Fisher Scientific NP0323BOX) for 3 h at constant voltage of 90 V. After gel electrophoresis, proteins were transferred onto Immobilon-P PVDF membranes (Millipore IPVH00010) for 90 min at constant current of 0.4A. Membranes were blocked with 1x Tris Buffered Saline (TBS) with 1% Casein (BioRad, #1610782) for 1 h at room temperature in gentle shaking, and then incubated at 4°C overnight with the primary antibody in blocking buffer, rabbit anti-Tyro3 (Cell signaling 5585S) at a dilution of 1:2000, mouse anti-PSD95 (Thermo Fisher Scientific MA1-046) at a dilution of 1:2000, mouse anti-GluA2 (Millipore MAB397) at a dilution of 1:5000, rabbit anti-synaptophysin (Abcam ab16659), or mouse anti-GAPDH (Santa Cruz SC-32233) at a dilution of 1:10,000. After washing with TBS buffer containing 0.1% Tween-20, membranes were incubated with secondary antibody for 3 h at room temperature (Peroxidase AffiniPure Goat Anti-Rabbit IgG, Jackson Immunoresearch 111–036-047 or 115–006-072). After washing with TBS buffer containing 0.1% Tween-20, membranes were incubated in chemiluminescent substrate (SuperSignal, ThermoFisher 34,580) and the chemiluminescent signal was detected and recorded by exposure of the membrane to X-ray film.