Ab205587

Prepared protein samples (10 μg) were subject to 12% SDS-PAGE, followed by electro-transfer onto PVDF membranes. Then the membranes were blocked in 5% skimmed milk for 1 h. Antibodies against exosome markers CD9 (ab92726; Abcam, Cambridge, UK), CD81 (ab109201; Abcam) and calnexin (ab133615; Abcam) were diluted at a ratio of 1:1000. The inflammatory response of vascular tissue and VSMCs was detected using antibodies against NFATc3 (18222-1-AP, 1:1000 dilution; Proteintech, Wuhan, China) and IL-6 (DF6087, 1:500 dilution; Affinity Biosciences, Cincinnati, USA), IL-1β (DF6251, 1:500 dilution; Affinity Biosciences) and TNF-α (ab205587, 1:500 dilution; Abcam). The internal control anti-β-actin primary antibody (TA-09; Zhongshan Jinqiao Biotechnology, Beijing, China) was used at 1:1000 dilution. The PVDF membranes were incubated with the above primary antibodies at 4°C overnight, followed by incubation with HRP-conjugated goat anti-mouse IgG (ZB-2305, 1:4000; Zhongshan Jinqiao Biotechnology) or HRP-conjugated goat anti-rabbit IgG (ZB-2301, 1:4000; Zhongshan Jinqiao Biotechnology) secondary antibodies for 1 h at room temperature. After extensive wash, protein bands were visualized using ultra high sensitivity ECL kit (HY-K1005; MCE, New Jersey, USA), and images were obtained with a chemiluminescence gel imaging system (12003153; Bio-Rad, Hercules, USA).

The vaginal tissues were lysed and homogenized in RIPA buffer with phenylmethylsulfonyl fluoride (PMSF) and quantified with a BCA kit (Keygen Biotech, KGP902, China). The protein samples (30 μg/lane) were separated using SDS-PAGE (Beyotim, P0012A, China) and transferred to polyvinylidene difluoride membranes (PVDF) (Merck, IPVH00010, United States) after electrophoresis. The membranes were blocked with 5% skimmed milk for 3 h, followed by incubation with TNF-α (1:1,000, Abcam, ab205587, United States), IL-1β (1:1,000, Abcam, ab234437, United States), IL-6 (1:1,000, Abcam, ab229381, United States), Dectin-1 (1:1,000, Abcam, ab140039, United States), Syk (1:1,000, Cell Signaling Technology, 13198T, United States), PLCγ-2 (1:1,000, Cell Signaling Technology, 3872T, United States), CARD9 (1:500, Affinity, DF8387, China), NF-κB (1:1,000, Cell Signaling Technology, 8242T, USA) and β-actin (1:2,000, Affinity, AF7018, China) antibodies overnight at 4°C. Then, the membranes were incubated with an HRP-conjugated antibody (1:5,000, Affinity, S0001, China) at room temperature for 1 h. The membranes were visualized with a chemiluminescence (ECL) kit by a Tanon 5200 system (Shanghai, China). ImageJ software (Version 1.52a) was used to measure the protein bands based on that of β-actin.

Testis tissue were lysed in lysis buffer and sonicated. The protein concentration was determined using a BCA protein assay kit (MDL, China). Approximately 30 μg of protein from each sample was separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5% skimmed milk in TBST and incubated with primary antibodies overnight at 4°C. Antibodies obtained from Abcam (Cambridge, United Kingdom) were as follows: MAPK3/1 (ERK1/2) (ab184699), TNF-α (ab205587). Antibodies purchased from Cell Signaling Technology (Danvers, MA, United States) included those against Akt (2938S) and EGFR (2646S). GAPDH (MDL, China) was regarded as the internal reference. Membranes were incubated with the corresponding secondary antibody (MDL, China) for 1 h at room temperature and washed in TBST. Protein signals were detected using ECL Detection Reagent kit (MDL, China). Images were captured by ChemiDoc MP Imaging System (Bio-rad, United States).

Tissues were homogenized in RIPA buffer (150 mM sodium chloride, 1.0% NP-40 or Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate, 50 mM Tris, pH 8.0) (10% weight/volume). Tissue samples were separated on SDS-PAGE (10–12%), and transferred onto a polyvinylidene difluoride membrane. Blots were incubated with the following primary antibodies: BDNF (1:250), Abcam, Cat. No. ab203573, RRID: AB_2631315), TLR4 (1:1500), Santa Cruz, sc-293072, RRID: AB_10611320), TLR2 (1:1000), Santa Cruz, sc-21760, NRF2 (1:5000), Santa Cruz, sc-722, TNFα (1:10,000), Abcam, Cat No ab205587, occludin (1:50,000), Abcam, Cat. No ab167161, p-NF-κB (1:10,000), Abcam, Cat No ab86299. Membranes were washed with TBS-T (TBS + 0.05% Tween20), and then incubated with a secondary antibody linked to horseradish peroxidase. As a loading control, α-actin (1:1000) or GAPDH (1:50,000) was used. Immunoreactive bands were visualized using Chemidoc (Bio rad XRS + SYSTEM). The western blots were performed at least 3 times using independent blots. Densitometry analysis was performed using NIH Image J software. Values were normalized with actin or GAPDH and expressed as fold increase.

The ipsilateral cerebral hemisphere, rat leukocytes, and cultured cells were homogenized in a lysis buffer containing phosphatase and protease inhibitors. The venous blood samples were obtained 24 h after MCAO surgery, and the leukocytes were separated as previously described. We used the following primary antibodies: anti‐interleukin 1 beta (IL‐1β) (Catalogue, AF‐501‐NA), anti‐tumor necrosis factor‐alpha (TNF‐α) (Abcam, ab205587), anti‐matrix metallopeptidase 9 (MMP‐9) (Abcam, ab76003), anti‐zonula occludens‐1 (ZO‐1) (Arigo, ARG55738), anti‐occludin (Abcam, ab216327), anti‐caspase‐3 (Abcam, ab13847; Affinity Biosciences, AF7022), anti‐C1QTNF6 (Abcam, ab36900), and anti‐β‐actin (Abcam, ab20272). Proteins were detected by incubation with horseradish peroxidase‐conjugated secondary antibodies (Santa Cruz Biotechnology) for 60 min at room temperature, using an enhanced chemiluminescence kit (Millipore). Gray values of the protein bands were analyzed using AlphaEase FC software (Alpha Innotech).

The cells were extracted with radio-immunoprecipitation assay (RIPA) lysis buffer supplemented with Protease Inhibitor Cocktail, and total protein was obtained for subsequent analysis. The concentration of protein was determined using bicinchoninic acid (BCA) method, while protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane for labeling. After blocking with 5% nonfat milk for 1 hour, the membranes were incubated with primary antibodies against Bax (1: 1000, ab32503, Abcam, UK), Caspase-3 (1: 1000, ab184787, Abcam, UK), Bcl-2 (1: 1000, ab194583, Abcam, UK), IL-6 (1: 1000, ab9324, Abcam, UK), IL-1β (1: 1000, ab254360, Abcam, UK), TNF-α (1: 1000, ab205587, Abcam, UK), TLR2 (1: 1000, ab209217, Abcam, UK), TLR4 (1: 1000, ab22048, Abcam, UK), NLRP3 (1: 1000, ab263899, Abcam, UK), Caspase-1 (1: 1000, ab 286,125, Abcam, UK) and β-actin (1: 5000, ab8227, Abcam, UK) at 4°C overnight. The membranes were washed and incubated with the secondary antibodies at room temperature for 2 h. An enhanced chemiluminescence detection system (Thermo Scientific, MA, USA) was finally used to determine the emission of the membrane. The western blot results were analyzed by Image J (Image J 1.46, NIH, Bethesda, MA, USA).