The following primary antibodies were used for western blotting: anti-ASC (ab180799), anti-caspase-1 (ab179515), anti-NLRP3 (ab263899), anti-gasdermin D (ab219800), anti-IL-1β (ab254360), anti-IL-18 (ab223293) (all purchased from Abcam, Cambridge, MA, USA), anti-F4/80 (28463–1-AP), anti-ZO-1 (21773–1-AP), anti-E-cadherin (20874–1-AP), and anti-AIM2 (66902–1-Ig) (all purchased from Proteintech). FITC goat anti-mouse IgG (H + L) and Cy3 goat anti-rabbit IgG (H + L) (A0516) were purchased from Beyotime Biotechnology (Haimen, China) and used for immunofluorescence analyses.
Ab180799
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab180799 is a lab equipment product offered by Abcam. It is a tool designed for scientific research purposes, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information from the manufacturer would be required to give a more comprehensive overview of the product.
Automatically generated - may contain errors
Lab products found in correlation
Ab263899, by Abcam (4 mentions)
Ab214185, by Abcam (4 mentions)
Ab179515, by Abcam (3 mentions)
Ab16502, by Abcam (3 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Ab254360, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab13243, by Abcam (2 mentions)
Ab93015, by Abcam (2 mentions)
Ab76302, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ab8245, by Abcam (2 mentions)
Cy3 goat anti rabbit igg h l a0516, by Beyotime (1 mentions)
Fitc goat anti mouse igg h l, by Beyotime (1 mentions)
Bca kit, by Solarbio (1 mentions)
Enhanced chemi luminescence (ecl), by Beyotime (1 mentions)
Ab7778, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab270993, by Abcam (1 mentions)
17 protocols using ab180799
1
Inflammasome Pathway Protein Detection
2
Western Blot Analysis of Protein Markers
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The target cell protein was obtained by RIPA method, and the protein concentration was determined by BCA kit (Solarbio, Beijing, China). Then, prepared protein was taken for gel electrophoresis separation, and the isolated protein was electrically transferred to PVDF membrane. After blocked with 5% skim milk, the PVDF membrane was subjected to primary antibodies: connective tissue growth factor (CTGF, #ab6992, 1:1000, Abcam, Cambridge, UK), collagen I (#ab270993, 1:1000, Abcam, Cambridge, UK), collagen III (#ab7778, 1:1000, Abcam, Cambridge, UK), Bcl-2 (#ab196495, 1:1000, Abcam, Cambridge, UK), Bax (#ab32503, 1:1000, Abcam, Cambridge, UK), NOD-like receptor protein 3 (NLRP3, #ab263899, 1:1000, Abcam, Cambridge, UK), ASC (rat: #ab180799, 1:1000, Abcam, Cambridge, UK; mouse: #67824S, Cell Signaling, Danvers, MA), caspase-1 (#83383, 1:1000, Cell Signaling, Danvers, MA), IL-β (#ab254360, 1:1000, Abcam, Cambridge, UK), IL-18 (#ab191860, 1:1000, Abcam, Cambridge, UK) and GAPDH (#ab9485, 1:1000, Abcam, Cambridge, UK). The next day, the PVDF membrane was incubated with secondary antibody (#ab288151, 1:5000, Abcam, Cambridge, UK) at room temperature. An enhanced chemiluminescence kit (ECL, #P0018S, Beyotime Biotechnology, Beijing, China) was added and exposed in the gel imaging system. The protein content was analysed using Quantity-One software (Bio-Rad, Hercules, CA).
3
Protein Extraction and Analysis from Cardiac Tissues
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Total proteins were extracted from cardiac tissues and H9c2 cells by radioimmunoprecipitation assay lysis buffer (Beyotime) and measured through a bicinchoninic acid protein assay kit (Beyotime). Protein samples (20 μg) were isolated by 10% SDS-PAGE (Beyotime) and transferred into polyvinylidene fluoride (PVDF; Beyotime) membranes. PVDF membranes were then treated with 5% skimmed milk/TBST for 1 h. Subsequently, the membranes were treated with primary antibodies overnight at 4 °C and secondary antibody for another 2 h at ambient temperature. The primary antibodies were as follows: anti-interleukin (IL)-1β (ab254360; 1:1000, Abcam), anti-nucleotide-binding oligomerization domain (NLRP3; ab263899; 1:1000, Abcam), anti-GSDMD-N (DF13758; 1:1000, Affinity), anti-caspase-1 (ab286125; 1 μg/mL, Abcam), anti-ASC (ab180799; 1:1000, Abcam), anti-transforming growth factor (TGF-β1; ab215715; 1:1000, Abcam), and anti-GAPDH (ab181602; 1:1000, Abcam). GAPDH was used as an internal reference. Finally, protein bands were visualized with an electrochemical luminescence on a Tanon 5200 machine (Tanon, Shanghai, China). Protein band intensities were detected with the Image J software (NIH, MD, USA).
4
Oxymatrine Modulates NLRP3 Inflammasome in INS-1 Cells
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The protein levels were analyzed by western blotting. The INS-1 cells were treated as follows: control; HG + PA; HG + PA + oxymatrine (1 μM); HG + PA + oxymatrine (10 μM). The cells were cultured in 6-well plates (4 × 105 cells/ml) for 24 h. In brief, the cell lysates were electrophoresed and transferred onto polyvinylidene fluoride membranes. Then, the membranes were incubated with the following primary antibodies (diluted in Tris-buffered saline with 0.1% Tween-20 [TBST] buffer): anti-NLRP3 antibody (ab214185, 1:1,000), anti-IL-1β antibody (ab205924, 1:1,000), anti-NF-κB p65 antibody (ab16502, 1:2,000), anti-Nrf2 antibody (ab89443, 1:500), anti-HO-1 antibody (ab13243, 1:2,000), anti-Keap1 antibody (ab119403, 1:1,000), anti-ASC antibody (ab180799, 1:2,000), and β-actin antibody (ab8226, 1:1,000) from Abcam (Cambridge, UK); anti-Gsdmd antibody (93709, 1:1,000) and anti-Histone H3 antibody (4499, 1:2,000) from Cell Signaling Technology (Boston, MA, USA); and anti-caspase-1 antibody (NBP1-45433, 1:1,000) from Novus Biologicals (Littleton, CO, USA). After washing, the membranes were incubated with the appropriate secondary antibody (diluted in TBST buffer) from Abcam (1:5,000, Cambridge, UK). Image Pro Plus 6.0 software (Media Cybernetics, Houston, TX, USA) was used to determine the intensity of the protein bands.
5
Western Blot Analysis of Apoptosis-related Proteins
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The specific steps of the western blot assay were performed, as previously described [22 (link), 33 (link)] with the following antibodies: anti-Caspase-1 (ab179515), anti-GSDMD (ab219800), anti-GSDMD-N (ab215203), anti-ASC (ab180799) and anti-AIM2 (ab93015) (Abcam, Cambridge, UK); anti-γH2AX (9718) (CST, Danvers, MA, USA); anti-PIF1 (19006-1-AP), anti-BRCA1 (22362-1-AP), anti-GAPDH (60004-1-Ig), and the secondary antibodies (Proteintech, Wuhan, China). Blots were conjugated with the chemiluminescent horseradish peroxidase substrate (Millipore), visualized, and quantified by Quantity 5.2 (Bio-Rad) according to the manufacturers’ instructions.
6
Protein Extraction and Western Blot Analysis
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Total proteins were extracted from colonic tissues and LGG-EVs using RIPA buffer containing protease and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). The equal quantity of protein was loaded into gels, and then transferred onto PVDF membranes (Thermo Fisher Scientific). Proteins were quantified by Image J software (NIH, Bethesda, MD). Antibodies against TSG101 (ab225877), p65 (ab16502), p-p65 (ab76302), NLRP3 (ab210491), ASC (ab180799) were purchased from Abcam (Cambridge, UK). β-actin (GB12001) was purchased from Servicebio (Wuhan servicebio technology CO., LTD, Wuhan, China).
7
Immunofluorescence Imaging of ASC and HBx
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HL7702 cells were plated into 24-well plates and grown to 50% confluence. Following plasmid transfection and H2O2 induction, immunofluorescence was performed. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton X-100 for 15 min. Cells were then blocked with 10% donkey serum for 60 min, incubated with primary antibodies overnight at 4 °C, and immunostained with secondary antibodies for 60 min at 23–25 °C. Primary antibodies specific for ASC (#ab180799; Abcam, Cambridge, MA, USA) and HBx (#ab235; Abcam, Cambridge, MA, USA) were used. The secondary donkey anti-mouse IgG (H + L), Alexa Fluor 488 (#A-21202; Invitrogen, Carlsbad, CA, USA), donkey anti-rabbit IgG (H + L), and Alexa Fluor 555 (#A-31572; Invitrogen, Carlsbad, CA, USA) antibodies were used. Finally, cells were observed using fluorescence confocal microscopy (Zeiss FM780, Jena, Germany). Pictures were taken under 63 × oil microscope and excitation light wavelengths of 405 nm, 488 nm, and 594 nm were selected for layer scanning with a scanning interval of 0.35 um/layer. The fluorescence excitation displayed blue, green, and red, respectively.
8
Western Blot Analysis of Oxidative Stress and Inflammation
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Cells were firstly lysed in RIPA buffer (Sigma-Aldrich, USA) and protein concentrations of each homogenate were measured using a commercial BCA protein assay Kit (Thermo Fisher Scientific, USA). 30 μg of proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was then blocked in 5% skimmed milk in TBST, and stained overnight at 4 °C with primary antibodies against Nrf2 (1:1000, ab137550, Abcam), HO-1 (1:2000, ab13243, Abcam), eNOS (1:5000, ab76198, Abcam), NQO1 (1:10,000, ab80588, Abcam), caspase-1 (1:1000, ab238979, Abcam), NLRP3 (1:1000, ab263899, Abcam), ASC (1:1000, ab180799, Abcam), HMGB1 (1:10,000, ab79823, Abcam), or β-actin (1:5000, ab115777, Abcam). Samples were then stained with goat anti-rabbit or goat anti-mouse IgG secondary antibody (1:10,000, ab205718/ab205719, Abcam) at room temperature for 2 h. Bands were developed using chemiluminescence substance (Thermo Fisher Scientific, USA). The proteins were quantified using Quantity One software (Bio-Rad, USA).
9
Western Blot Analysis of Inflammatory Signaling
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Cellular lysates were obtained by lysis of the cell strains in RIPA buffer. After the concentration was determined, the proteins were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene diflouride (0.45 µm) membrane. The membrane was then blocked with BSA (5%) at 25 °C for 1 h. Using the intended antibodies, the membrane was cultured at 4 °C for approximately 16 h. Thereafter, the secondary antibodies were applied (4 °C; 1 h). The antibodies used in this experiment were: TLR9 antibody (ab134368, Abcam), P-NF-kB antibody (ab264271, Abcam), NF-kB antibody (ab16502, Abcam), NLRP3 antibody (ab4207, Abcam), Caspase-1 antibody (ab138483, Abcam), IL-1 antibody (ab9722, Abcam), PYCARD antibody (ab180799, Abcam), LaminB1 antibody (ab16048, Abcam), Collagen IV antibody (ab19808, Abcam), Fibronectin antibody (ab2413, Abcam), Bak antibody (ab104124, Abcam), Bax antibody (ab53154, Abcam), Bcl-xl antibody (ab32370, Abcam), Bcl-2 antibody (ab59348, Abcam), and Actin antibody (ab8227, Abcam). Based on the C-DiGit® Blot Scanner, a strengthened chemiluminescent reaction (SuperSignal®West Femto Maximum Sensitivity Substrate Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA) was conducted in order to visualize the blots.
10
Western Blot for NLRP3 Inflammasome Proteins
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The liver tissues in each group were ground, and the hepatic cells in each group were harvested and washed with PBS. Total proteins were extracted using a protein extraction kit (BestBio; BB-3101), and the protein concentration in each extract was determined using the bicinchoninic acid (BCA) method. Next, a 20 μg aliquot of total protein from each extract was separated by 10% SDS-PAGE performed at 120 V. The separated protein bands were electrophoretically (200 mA for 90 mins) transferred onto PVDF membranes (Roche, Basal Switzerland, cat. no. 3010040001), which were subsequently blocked with 5% powdered skim milk. Next, the PVDF membranes were incubated with primary antibodies at 4°C overnight. After washing, the PVDF membranes were soaked with an HRP-labeled secondary antibody for 1 h and the immunostained protein bands were detected using the ECL chemiluminescence reagent (Millipore; KLS0500). The primary antibodies used in the study were as follows: NLRP3 (1 : 1000, Abcam, Cambridge, UK, ab214185), ASC (1 : 1000, Abcam, ab180799), and caspase 1 (1 : 1000, Abcam, ab62698).
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