Phosphatase inhibitor

The total protein was extracted from brain tissues, then homogenized in RIPA lysis buffer including protease inhibitor (Solarbio, China) and phosphatase inhibitor (Solarbio, China) for 30 min on ice. Protein of supernatant was collected after centrifugation for 15 min (13,600 ×g, 4°C). Total protein was detected using a bicinchoninic acid (BCA) kit (Thermo Scientific, USA). Brain lysates were electrophoresed on 10% SDS-PAGE, transferred to nitrocellulose membrane, then blocked with tris-buffered saline with Tween 20 (TBST) solution containing 3%BSA. Membranes were probed with primary antibodies for MyD88 (Abcam, USA), TRAF6 (Abcam, USA), NF-κB p65 (Abcam), Nrf2 (Bioss, China), HO-1 (Bioss, China), GAPDH (Abcam) and β-actin (Abcam) overnight at 4°C, and washed with TBST 3 times for 10 min. Membranes were treated with HRP-conjugated anti-rabbit antibody (Bioss, China) the next day. Protein bands were analyzed using enhanced chemiluminescence and calculated using ImageJ software.

Cells were lysed in cold RIPA buffer (Solarbio Science & Technology, Beijing, China) supplemented with 1 mM PMSF (Solarbio Science & Technology, Beijing, China) and 10 mM phosphatase inhibitor (Solarbio Science & Technology, China). The cell lysates were centrifuged at 12,000× g for 10 min at 4 °C to remove the precipitate. Protein concentrations of samples were examined using a BCA protein assay kit (Beyotime Biotechnology, Nantong, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in TBST for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. After primary antibody incubation, the membranes were rinsed three times in TBST followed by incubation with HRP-labeled goat anti-rabbit IgG antibody (Servicebio, Wuhan, China) for 1 h at room temperature. β-Actin was used as a loading control.

Western blotting assay was performed as described (27 (link), 28 (link)). Briefly, total protein was extracted using RIPA lysis buffer (Beyotime) with 1% PMSF (Beyotime) and 1% phosphatase inhibitor (Solarbio). Protein concentration was determined using a bicinchoninic acid (BCA) Assay Kit (Beyotime). Protein extracts were resolved through 10% SDS-PAGE after denaturation, transferred to PVDF membranes (0.22µm, Millipore) and probed with rabbit anti-H3Cit (ab5103, 1:1000, Abcam) and rabbit anti-H3 (ab1791, 1:5000, Abcam) primary antibodies at 4°C overnight. The membranes were then incubated in the second antibody for 1 hour at room temperature. An enhanced chemiluminescence (Millipore) were used for HRP detection. Quantitative analysis of Western blotting bands was performed using Image J (NIH).

Neurons were lysed at 30 days with radioimmunoprecipitation assay (RIPA) lysis (Solarbio, Cat. #R0010, China), PMSF (Solarbio, Cat. # P0100, China), and phosphatase inhibitor (Solarbio, China) in proportions of 100:1:1, respectively. A BCA Protein Assay Kit (KeyGEN, Cat. #KGP902, China) was used for protein quantification. The proteins were used with 4–20% sodium dodecyl sulfate–polyacrylamide gel for electrophoresis and were transferred into the PVDF membrane (Merck Millipore, Cat. #ISEQ00010, Germany). They were then blocked with 5% nonfat milk for 90 min and incubated overnight with the primary antibody at 4°C. The following day, they were incubated with the secondary antibody. After that, an Omni-ECL™ Enhanced Pico Light Chemiluminescence Kit (EpiZyme, Cat. #SQ101, China) was added to capture the protein with a ChemiDoc™ MP Image System (Bio-RAD, Universal Hood III). The information about the antibodies is as follows: Kv4.3 (1:200; Affinity Biosciences); activating transcription factor 4 (ATF4) (1:200; Cell Signaling Technology); and C/EBP homologous protein (Bushart et al., 2018 (link)) (1:200, Proteintech). Goat anti-mouse IgG (H+L) -HRP (1:10,000; Bioworld) and goat anti-rabbit IgG (H+L) HRP (1:10,000; Bioworld) were used for Western blotting.

Recombinant human TNF-α (210-TA/CF) was purchased from R&D Systems (Minneapolis, MN, USA). Ammonium pyrrolidinedithiocarbamate (PDTC) were purchased from Abcam (Cambridge, UK). TPCA-1 (GW683965) was purchased from Selleck (Shanghai, China). Lipofectamine3000, Bio-16-UTP, Streptavidin agarose beads, and TRIzol reagent were purchased from Thermo Fisher Scientific Life Sciences (Waltham, MA, USA). Actinomycin D (Act D) was purchased from Sigma-Aldrich (St Louis, MO, USA). Protease Inhibitor Cocktail tablets were purchased from Roche (Basel, Switzerland). Phosphatase inhibitor was purchased from Solarbio Science & Technology Co. (Beijing, China). The fluorescence-labeled probes for lncRNA fluorescence in situ hybridization (FISH) assays, miRNA mimics/inhibitors and miRNA primers were obtained from RiboBio Co. Ltd (Guangzhou, China).

Amylase (C016-1–1), Trypsin (A080-2–2), and Lipase (A054-2–1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRIzol reagent was obtained from Tiangen Biotech Co., Ltd. (Beijing, China). Reverse transcription kit from TaKaRa (Japan), 2 × SYBR Green qPCR Master Mix from Bimake (Houston, USA), RIPA cell lysate, protease inhibitor and phosphatase inhibitor were purchased from Solarbio (Beijing, China). β-actin Monoclonal Antibody was purchased from Immunoway Bitechnology company (Jiangsu, China). ZO-1(ab96587) was obtained from Abcam (Cambridge, MA, USA). Occludin (bs-10011R) was obtained from Bioss (Wuhan, China). Claudin-1 (13,050–1-AP) was obtained from Proteintech (Wuhan, China) and a DAB kit was purchased from CWBIO (Beijing, China). Fusarium graminearum ACCC 37,687 was provided by Professor Yang Xiaojun, College of Animal Science and Technology, Northwest A&F University. AA broilers were purchased from Shanxi Elephant Farming and animal husbandry group (China).

TU686/SCC25/WSU-HN30 cells were treated with RIPA lysate buffer containing a protease inhibitor (Solarbio, Beijing, China) and a phosphatase inhibitor (Solarbio). Concentrations of proteins within supernatants were determined using a BCA kit (Dowobio, Shanghai, China). Proteins (20 μl per lane) and Marker (5 μl per lane) ,DW1106 (Dowobio, Shanghai, China) were loaded into wells of SDS-PAGE gels followed by electrophoresis to separate proteins by molecular weight (8–10% SDS-PAGE separation gel and a 5% SDS-PAGE concentrated gel). Next, proteins were transferred to polyvinylidene flfluoride membranes (PVDF, Solarbio) using a wet transfer method. PVDF membranes bound to proteins were blocked by immersion in 5% (w/v) bovine serum albumin (Solarbio), then membranes were probed with antibodies anti-Amphigireegulin, ER1903-67; anti-Urokinase Recombinant, ET1703-26, anti-Amyloid Beta A4 Precursor(APP), 1007–5; anti-Caveolin-2, ET1607-15 (HUABIO, Hangzhou, China) as dilutions of 1:1500, GAPDH, ab181602(Dowobio, Shanghai, China), as dilutions of 1:8000,followed by incubation at 4° C overnight. Next, membranes were incubated with secondary antibody (HRP) at room temperature for 1 h. An enhanced ECL,BL523B(Biosharp, AnHui,China) reagent is added to the film, developed after exposure with blue film, fixed, and scanned by a scanner for analysis.