Ab243091

Manufactured by Abcam

Sourced in United Kingdom, United States

Ab243091 is a laboratory product offered by Abcam. It is designed for use in research applications. The core function of this product is to provide a tool for researchers in their scientific investigations, though further details on its specific applications or intended use are not available.

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Lab products found in correlation

Ab263899, by Abcam (6 mentions) Ab8226, by Abcam (4 mentions) Ab254360, by Abcam (4 mentions) Ab216995, by Abcam (2 mentions) Ab215203, by Abcam (2 mentions) Ab207802, by Abcam (2 mentions) Ab179515, by Abcam (2 mentions) Ab209845, by Abcam (2 mentions) Rnascope kit, by Advanced Cell Diagnostics (1 mentions) Anti il 1β antibody, by Cell Signaling Technology (1 mentions) Haf007, by R&D Systems (1 mentions) Anti cd66b antibodies, by BioLegend (1 mentions) Axioscope ai, by Zeiss (1 mentions) Image lab software, by Bio-Rad (1 mentions) Ab129002, by Abcam (1 mentions) Ab32360, by Abcam (1 mentions) Ag 20b 0014 c100, by Adipogen (1 mentions) Enhanced chemi luminescence (ecl), by Advansta (1 mentions) Sa00001 1, by Proteintech (1 mentions) Sa00001 2, by Proteintech (1 mentions)

11 protocols using ab243091

Immunohistochemistry and RNA in situ Hybridization of Inflammatory Markers

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For immunohistochemistry (IHC), sectioned formalin-fixed paraffin-embedded biopsy samples were deparaffinized and rehydrated, which was followed by heat-induced and enzymatic antigen retrieval. Subsequently, sections were blocked and incubated at 4 °C overnight with the following primary antibodies: anti-IL-1β antibody (1:100, 12242; Cell Signaling Technology, Inc., Danvers, MA, USA) or anti-IL-18 antibody (1:1000, ab243091; Abcam, Cambridge, UK). Thereafter, biotinated or horseradish peroxidase-conjugated secondary antibodies (HAF007; R&D Systems, Inc., Minneapolis, MN, USA) were used for detection. Anti-CD66b antibodies (1:100, 3929023, Biolegend, San Diego, CA, USA) were used for the detection of neutrophils. Images were captured using Axio Scope AI (ZEISS, Oberkochen, Germany).
For RNA in situ hybridization, the RNAScope Kit (Advanced Cell Diagnostics, Newark, CA, USA) was used to measure the mRNA expression in paraffin-embedded sections.

Furuta Y., Gushima R., Naoe H., Honda M., Tsuruta Y., Nagaoka K., Watanabe T., Tateyama M., Fujimoto N., Hirata S., Miyagawa E., Sakata K., Mizuhashi Y., Iwakura M., Murai M., Matsuoka M., Komohara Y, & Tanaka Y. (2023). Possible Association of Mutations in the MEFV Gene with the Intestinal Phenotype of Behçet’s Disease and Refractoriness to Treatment. Journal of Clinical Medicine, 12(9), 3131.

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Protein Expression Analysis in THP-1 Cells

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Total protein was extracted from THP-1 cells using protein lysis solution. After protein quantification, electrophoresis and transmembrane, the protein samples were incubated with primary antibodies against vinculin (1:10,000 dilution; ab129002; Abcam, Cambridge, UK), AKT (1:1,000 dilution; 9271; CST, Danvers, MA, USA), phospho-AKT (1:1,000 dilution; 2211; CST), IL-1β (1:1,000 dilution; 12242; CST), IL-18 (1:1,000 dilution; ab243091; Abcam), nucleotide‐binding oligomerization domain (NOD)‐like receptor protein 3 (NLRP3; 1:1,000 dilution; AG-20B-0014-C100; AdipoGen, San Diego, CA, USA), caspase‐1 (1:10,000 dilution; ab108362; Abcam), NF‐κB p65 (1:1,000 dilution; AB32360; Abcam), or phospho‐NF‐κB p65 (1:1,000 dilution; 3033; CST) overnight at 4°C. Secondary antibodies conjugated with horseradish peroxidase (1:5,000 dilution; SA00001-1; SA00001-2; Proteintech, Rosemont, PA, USA) were incubated at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence kit (ECL; Advansta, Menlo Park, CA, USA). Data acquisition and analysis were performed on a gel image analyzing system equipped with Image Lab Software (Bio‐Rad, Hercules, CA, USA).

Liu Z., Li J., Lin S., Wu Y., He D, & Qu P. (2021). PI3K regulates the activation of NLRP3 inflammasome in atherosclerosis through part-dependent AKT signaling pathway. Experimental Animals, 70(4), 488-497.

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Inflammatory Pathway Protein Analysis

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RIPA lysis buffer (Bolingkewei, Beijing, China) was applied to homogenize the tissues. The whole protein was extracted and quantified using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL, USA). The protein (35 μg/lane) was isolated through 12% SDS-PAGE gels, followed by transferring onto polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). After 1 h incubation with 5% defatted milk, membranes were probed at 4°C overnight against the following antibodies: NLRP3 (ab263899, Abcam), IL-18 (ab243091, Abcam), cleaved caspase-1 (orb126550, Biorbyt), IL-1β (ab216995, Abcam), N-cadherin (ab76011, Abcam), E-cadherin (ab40772, Abcam), fibronectin (ab2413, Abcam), vimentin (ab92547, Abcam), and β-actin (ab8226, Abcam). On the following day, membranes were exposed to goat anti-mouse (ab6789, Abcam) or goat anti-rabbit (ab6721, Abcam) HRP-conjugated secondary antibodies for 2 h at room temperature. Eventually, the signals were developed with an enhanced chemiluminescence system (Millipore, USA), followed by quantification with ImageJ software (NIH, Bethesda, Maryland, USA).

Li W., Zhao X., Zhang R., Xie J, & Zhang G. (2023). Silencing of NLRP3 Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin. Mediators of Inflammation, 2023, 7700673.

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Inflammasome Protein Expression Analysis

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Proteins were extracted and quantified using radio immunoprecipitation assay (RIPA) buffer (Biovision, American) and a protein concentration detection kit (absin, Shanghai, China). The prepared protein was separated by polyacrylamide-sodium dodecyl sulfate (SDS) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Roche, Switzerland). Next, the membranes were incubated overnight at 4 ℃ with polyclonal antibodies: cleaved-caspase-1 (#4199T, 1:1,000, Cell Signaling Technology, USA), NOD-like receptor family pyrin domain containing 3 (NLRP3) (ab263899, 1:1,000, Abcam, UK), apoptosis-associated speck-like protein containing a CARD (ASC) (#13833S, 1:1,000, Cell Signaling Technology, USA), gasdermin D (GSDMD)-N (ab215203, 1:1,000, Abcam, UK), interleukin (IL)-1β (ab216995, 1:1,000, Abcam, UK), IL-18 (ab243091, 1:1,000, Abcam, UK), and β-actin (ab8226, 1:1,000, Abcam, UK). After washing with Tris-HCl buffered saline with 0.1% (v/v) Tween 20 (TBST), the membranes were incubated with a secondary antibody, and protein expression intensity was determined by enhanced chemiluminescent (ECL) kit (Beyotime, Beijing, China).

Kong N., Xu Q., Cui W., Feng X, & Gao H. (2022). PCSK9 inhibitor inclisiran for treating atherosclerosis via regulation of endothelial cell pyroptosis. Annals of Translational Medicine, 10(22), 1205.

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Western Blot Analysis of Synaptic and Inflammatory Proteins

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The tissues were homogenized in ice-cold RIPA lysis buffer (Beyotime, Shanghai, China) and supplemented with complete EDTA-free protease inhibitor cocktail and PhosSTOP Phosphatase Inhibitor. The protein samples were mixed with 5 × dual color protein loading buffer (Fudebio, Hangzhou, China) and boiled at 98 °C for 10 min. Equal amounts of protein (30 μg/lane) were loaded on a SDS-PAGE gel at a constant voltage and then transferred to a polyvinylidene difluoride membrane (Millipore, MA, USA). After being blocked and incubated with the primary and secondary antibodies, the bands were visualized using a chemiluminescence image analysis system (Tanon, Shanghai, China) with FDbioFemto ECL (Fudebio, Hangzhou, China). The band intensities were quantified using Gel-Pro Analyzer software (Media Cybernetics, Maryland, USA). The primary antibodies were included the following: synaptosomal associated proteins 25 (SNAP-25; Abcam, ab109105), postsynaptic density proteins 95 (PSD-95; CST, 3450), Ibα1 (Abcam, ab178846), TNF-α (Abcam, ab215188), IL-6 (Abcam, ab233706), NLRP3 (Abcam, ab263899), IL-18(Abcam, ab243091), IL-1β (Abcam, ab254360), caspase-1 (Abcam, ab207802), TLR4 (Abcam, ab22048), myeloid differentiation factor 88 (MyD88; Abcam, ab133739), NF-κB p65 (Abcam, ab32536), GAPDH (ABclonal, AC033), ZO-1 (ABclonal, A11417), occludin (ABclonal, A12621) and β-actin (ABclonal, AC026).

He Q., Zhang L.L., Li D., Wu J., Guo Y.X., Fan J., Wu Q., Wang H.P., Wan Z., Xu J.Y, & Qin L.Q. (2023). Lactoferrin alleviates Western diet-induced cognitive impairment through the microbiome-gut-brain axis. Current Research in Food Science, 7, 100533.

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Immunoblotting analysis of inflammasome proteins

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The HLMVECs were lysed in radioimmunoprecipitation assay lysis buffer containing protease and phosphatase inhibitors (Solarbio). Protein concentration in the cell lysate was determined using a bicinchoninic acid (BCA) protein quantification kit. Protein samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk for 1 h and incubated with primary antibody at 4 °C overnight. Primary antibodies used in this assay were as follows: GSDMD (Abcam, ab210070), GSDMD-N (Abcam, ab215203), Caspase1 (Abcam, ab207802), Caspase1 p20 (CST, 4199T), IL-1β (CST, 83186), IL-18 (Abcam, ab243091), NLRP3 (Abcam, ab263899), NOX4 (Abcam, ab133303), TRX (abcam, ab133524), TXNIP (CST, 14715). Protein signals were detected using SuperSignal West Pico substrate (Thermo Scientific, Rockford, IL, USA). Finally, the protein signals were quantified by ImageJ.

Yao C., Kong J., Xu F., Wang S., Wu S., Sun W, & Gao J. (2024). Heme-Inducing Endothelial Pyroptosis Plays a Key Role in Radiofrequency Ablation of Hepatic Hemangioma Leading to Systemic Inflammatory Response Syndrome. Journal of Inflammation Research, 17, 371-385.

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Regulation of NLRP3 and Mitophagy in Angiotensin II-Induced Cardiomyocyte Injury

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NLRP3 inflammasome-related proteins including NLRP3 (1:1000, no. ab263899, Abcam, USA), ASC (1:1000, no. ab283684, Abcam), pro-caspase-1(1:1000, no. ab179515, Abcam), caspase-1(1:1000, no. ab207802, Abcam), IL-1β (1:1000, no. ab254360, Abcam), IL-18 (1:1000, no. ab243091, Abcam), and mitophagy-related proteins including PINK-1 (1:1000, no. ab300623, Abcam), Parkin (1:2000, no. ab77924, Abcam), beclin-1(1:1000, no. ab302669, Abcam), LC3B (1:2000, no. ab192890, Abcam), p62 (no. ab207305, Abcam) were quantified by western blotting. β-actin as reference. Cultured H9C2 cells for 24 h with 1 μM AngII and 0, 0.3, 0.6, and 1.2 mM hirudin, respectively, PBS as a control. RIPA buffer (no. P0013K, Beyotime, China) was used for lysed samples. SDS-PAGE was used to separate lysates and proteins were transferred to polyvinylidene difluoride (PVDF) membranes. Then, the membranes were blocked with 5 % non-fat milk at room temperature for 1 h and incubated with primary antibodies at 4 °C. After overnight incubation, incubated with secondary antibody for 1 h. Detected the Protein expressions with high sensitivity ECL chemiluminescence detection kit (no. PK10002, Proteintech, China). Using ImageJ software (National Institutes of Health, Bethesda, MD).

Luo G., Chen L., Chen M., Mao L., Zeng Q., Zou Y., Xue J., Liu P., Wu Q., Yang S, & Liu M. (2023). Hirudin inhibit the formation of NLRP3 inflammasome in cardiomyocytes via suppressing oxidative stress and activating mitophagy. Heliyon, 10(1), e23077.

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Immunostaining of Inflammatory Markers

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Immunohistochemistry and immunofluorescence staining were performed as described previously [22] [23] [24] . Briefly, slides were incubated with anti-GSDMD reacted with mouse (ab209845, abcam) (1:50), anti-GSDMD reacted with human (ab155233, abcam) (1:50), anti-IL-18 (ab243091, abcam) (1:200), anti-IL-1β (ab254360, abcam) (1:200), anti-CD206 (ab64694, abcam) (1:200), anti-NOS2 (sc7271, Santa Cruz) (1:200), and anti-F4/80 (RT1212, HUABIO) (1:200) antibodies for immunofluorescence staining, respectively. The expression of α-SMA and collagen I was assessed by immunohistochemistry using anti-α-smooth muscle actin (α-SMA) antibody (1:200; Boster, Wuhan, China), and anti-collagen type I (1:100; Proteintech, Wuhan, China) antibodies. The slides were then processed with the appropriate secondary antibodies/horseradish peroxidase (1:200).

Wu J., Wang P., Xie X., Yang X., Tang S., Zhao J., Liu T., Wang J., Zhang J., Xia T, & Feng X. (2024). Gasdermin D silencing alleviates airway inflammation and remodeling in an ovalbumin-induced asthmatic mouse model. Cell death & disease, 15(6).

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Western Blot Antibody Validation Protocol

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Western blot was performed using standard protocol as previously described [28] . The following primary antibodies were used: anti-GSDMD (ab209845, abcam), anti-IL-18 (ab243091, abcam) (1:2000), anti-IL-1β (ab254360, abcam) (1:2000), anti-caspase-1 (ab180673, abcam) (1:1000), anti-caspase-11(ab138483, abcam) (1:1000), anti-Notch 4 (A-12) (sc-393893, Santa Cruz) (1:1000), anti-DTX4 (25222-1-AP, Proteintech, 1:100), and anti-β-actin (ab8226, abcam) (1:10000) antibodies. β-actin was used as an internal control.

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Quantitative Protein Analysis of Cell Signaling

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A549 or H1650 cells were lysed. Then, the proteins were quantified using a BCA assay (#23225, Thermo Scientific). Subsequently, an equal amount of protein samples was subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to 0.22 μm polyvinylidene difluoride membranes (PVDF, Millipore, HATF09025). The membranes were blocked with 5% BSA and incubated rabbit anti-PD-L1 (ab205921, Abcam, Cambridge, UK), anti-IL-18 (ab243091, Abcam), anti-p65 (ab32536, Abcam), anti-p-p65 (ab31624, Abcam), anti-METTL3 (ab195352, Abcam), anti-p-IκBα (#2859, Cell Signaling), anti-IκBα (#9242, Cell Signaling), anti-YTHDF2 (#80014, Cell Signaling), anti-METT16 (#17676, Cell Signaling), anti-WTAP (#56501, Cell Signaling), anti-FTO (#14386, Cell Signaling), anti-ALKBH5 (#80283, Cell Signaling), anti-METTL14 (#51104, Cell Signaling) and anti-α-SMA (#14968, Cell Signaling) antibodies overnight at 4°C.β-actin (ab8226, Abcam) was used as the endogenous control. On the second day, the membranes were then incubated with a secondary antibody (Jackson, 1:10000) for 1 h at room temperature followed by three 10 min washes in TBST. The bands were visualized using an enhanced chemiluminescence (ECL) Detection Kit (E-IR-R308, Elabscience). Data were quantified using the ImageJ Launcher software (National Institutes of Health).

Xu L., Li K., Li J., Xu F., Liang S., Kong Y, & Chen B. (2023). IL-18 serves as a main effector of CAF-derived METTL3 against immunosuppression of NSCLC via driving NF-κB pathway. Epigenetics, 18(1), 2265625.

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