Anti a20

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-A20 is a laboratory reagent that can be used to detect and quantify the A20 protein in various biological samples. A20 is a key regulator of inflammatory signaling pathways. This product can be used in applications such as Western blotting, immunoprecipitation, and ELISA to study the expression and function of A20 in cells and tissues.

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Lab products found in correlation

Nec 1, by Merck Group (2 mentions) Anti bim, by BD (2 mentions) Anti bclx, by BD (2 mentions) Anti bcl 2, by Merck Group (2 mentions) Anti ripk3, by Santa Cruz Biotechnology (2 mentions) Anti ripk1, by Cell Signaling Technology (2 mentions) Anti k63, by Merck Group (2 mentions) Dynabead protein a, by Thermo Fisher Scientific (2 mentions) Anti ubiquitin, by Santa Cruz Biotechnology (2 mentions) Anti bax, by Santa Cruz Biotechnology (2 mentions) Dynabeads m 270 epoxy, by Thermo Fisher Scientific (2 mentions) Celltiter glo luminescent cell viability assay, by Promega (2 mentions) β actin, by Merck Group (2 mentions) Anti ripk1, by BD (2 mentions) Anti ripk3, by ProSci (2 mentions) Anti ubiquitin, by Cell Signaling Technology (1 mentions) Anti usp14, by Cell Signaling Technology (1 mentions) Anti uchl3, by Cell Signaling Technology (1 mentions) Anti skp2, by Cell Signaling Technology (1 mentions) Anti stat5, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Anti-A20 antibody (3 citations)

7 protocols using anti a20

RIPK1-Mediated Necroptosis Signaling

Cited 1 time

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Stimulated T cells were incubated in lysis buffer. For signaling complex analyses, cell lysates were pre-cleared with Protein G beads prior to immunoprecipitation with anti-RIPK1 antibody. Antibodies and reagents used for immunoprecipitation and immunoblotting studies included: anti-RIPK1, anti-Bim, anti-Bclx, (BD Biosciences), nec1, anti-Bcl2, β-actin (Merck chemicals), anti-RIPK3 (ProSci Incorporated), anti-ubiquitin, anti-Bax, anti-RIPK3 (Santa Cruz Biotechnology), anti-RIPK1, anti-A20 (Cell signaling Technology), QVD, ZVAD (Enzo Life Science).
Signaling studies in MEFs were performed as described^{19 (link)}. RIPK1 immunoprecipitations were performed using anti-RIPK1 (Cell Signaling) and Dynabeads M270 Epoxy (Invitrogen). K63-ubiquitin immunoprecipitations were performed using anti-K63 (Millipore) and Dynabead Protein A (Invitrogen). Studies of RIPK3 mutants in A20^−/−Ripk3^−/− MEFs were performed by generating RIPK3 lysine mutants, introducing mutant RIPK3-encoding cDNAs into GFP expressing lentiviral constructs, and infecting A20^−/−Ripk3^−/− MEFs with these viruses. Productively infected cells were FACS-sorted to obtain pure populations of RIPK3-expressing cells prior to being tested in necroptosis assays. Cell survival of MEFs was quantitated using the CellTiter-Glo Luminescent Cell Viability Assay per manufacturer’s instructions (Promega).

Onizawa M., Oshima S., Schulze-Topphoff U., Oses-Prieto J.A., Lu T., Tavares R., Prodhomme T., Duong B., Whang M.I., Advincula R., Agelidis A., Barrera J., Wu H., Burlingame A., Malynn B.A., Zamvil S.S, & Ma A. (2015). The ubiquitin-modifying enzyme A20 restricts the ubiquitination of RIPK3 and protects cells from necroptosis. Nature immunology, 16(6), 618-627.

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Ubiquitin-mediated protein regulation assay

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Spautin-1 (S7888), Imatinib (S1026), and MG132 (S2619) were from Sellectchem (Houston, TX, USA). SKP2-C25 (M60136) was from Xcessbio Biosciences, Inc. (San Diego, CA). MTS reagent (G3582) was from Promega Corporation (Madison, WI, USA). Co-IP assay kit (14311D) was from Life Technologies (Carlsbad, CA). Antibodies: anti-Ubiquitin (#3936), anti-K63-Ubiquitin (#12930), anti-K48-Ubiquitin (#12805), anti-USP10 (#8501), anti-USP1 (#8033), anti-USP2 (#8036), anti-USP7 (#4833), anti-USP8 (#11832), anti-USP14 (#11931), anti-USP15 (#66310), anti-USP18 (#4813), anti-UCHL1 (#13179), anti-UCHL3 (#8141), anti-CYLD (#8462), anti-A20 (#5630), anti-SKP2 (#2652), anti-p27 (#3686), anti-FLAG (#14793), anti-HA (#3724), anti-phospho-c-Abl(Y245) (#2861), anti-c-Abl (#2862), anti-phospho-STAT5 (#9359), anti-STAT5 (#25656), anti-phospho-Crkl (#3181) and anti-Crkl (#38710) (Cell Signaling Technology, Beverly, MA, USA); anti-UCHL5 (ab124931), anti-USP13 (ab109264) (Abcam, Cambridge, MA); anti-GAPDH (BS60630) (Bioworld Technology, Inc., Louis Park, MN, USA).

Liao Y., Liu N., Xia X., Guo Z., Li Y., Jiang L., Zhou R., Tang D., Huang H, & Liu J. (2019). USP10 modulates the SKP2/Bcr-Abl axis via stabilizing SKP2 in chronic myeloid leukemia. Cell Discovery, 5, 24.

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Evaluating CD40-mediated Activation in U2OS and iDCs

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To evaluate CD40-mediated activation we analyzed p100 processing and TRAF1 induction in U2OS cells and iDCs by western blotting. For western blot, coculture experiments were performed in six well plates (10⁶ +10⁶ cells per well). For stimulation of iDCs 0.8 × 10⁶ cells per well were seeded also in 6-well plates and stimulated overnight. Cells were collected in ice-cold PBS by scraping with a rubber policeman. Cells were then washed twice with fresh ice-cold PBS, pelleted (5 min, 4°C, 4630 g) and resuspended in Laemmli buffer. Samples were sonicated for 25 seconds at 100% amplitude with a sonication probe (UP100H Ultrasonic Processor, Helscher, Germany), heated at 95°C for 5 min and subjected to SDS-PAGE separation. After transfer of proteins to a nitrocellulose membrane western blot analysis was performed with an anti-p100/p52 (#05–361, Millipore), anti-TRAF1 (#4715), anti-A20 (#5630, both Cell Signaling Technology Beverly, MA, USA), anti-β-actin (#A1978–200), anti-Flag (M2) (#F-3165, both Sigma Aldrich) and horseradish peroxidase (HRP)-conjugated polyclonal rabbit anti-mouse antibody (#P0260, Dako, Glostrup, Denmark) or HRP-coupled anti-rabbit antibody (#7074, Cell Signaling Technology Beverly, MA, USA). Finally, membranes were developed by chemiluminescence western blot detection using ECL solution.

Hesen N., Anany M., Freidel A., Baker M., Siegmund D., Zaitseva O., Wajant H, & Lang I. (2024). Genetically engineered IgG1 and nanobody oligomers acquire strong intrinsic CD40 agonism. Bioengineered, 15(1), 2302246.

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Protein Extraction and Western Blot Protocol

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Cells were centrifuged at 1200 rpm at 4 °C for 10 min to collect cells. Cell pellets were lysed with radioimmunoprecipitation assay (RIPA) buffer (Santa Cruz, Dallas, TX, USA) containing a proteinase inhibitor cocktail (Thermo Fisher Scientific) and incubated for 10 min on ice. The lysed cell pellet was centrifuged at 12,500 rpm at 4 °C for 10 min to isolate the proteins. The concentration of isolated proteins was measured using the bicinchoninic acid protein (BCA) assay. Proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidenedifluoride membrane with a 0.2 μm pore size (Bio-Rad, Hercules, CA, USA). The target proteins were detected and analyzed by immunoblotting using the primary antibodies diluted in 0.1% PBS-Tween buffer containing 1% bovine serum albumin and 0.02% sodium azide. These primary antibodies included anti-A20 (1:1000) (Cell Signaling, Danvers, MA, USA) and anti-GAPDH (1:2000) (Enzo Life Science, Farmingdale, NY, USA).

Choi S., Park H., Jung S., Kim E.K., Cho M.L., Min J.K., Moon S.J., Lee S.M., Cho J.H., Lee D.H, & Nam J.H. (2017). Therapeutic Effect of Exogenous Truncated IK Protein in Inflammatory Arthritis. International Journal of Molecular Sciences, 18(9), 1976.

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RIPK1-Mediated Necroptosis Signaling

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THP-1 Cells Immunoblotting Protocol

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THP-1 cells stimulated with LPS and/or MSF were harvested and lysed with 2× Laemmli buffer. Denatured proteins were separated by 12% SDS-PAGE in a Tris/glycine/SDS buffer (25 mM Tris, 250 mM glycine, 0.1% SDS) and transferred onto PVDF membranes (EMD Millipore, Burlington, MA, USA). Membranes were blocked with TBST (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) containing 5% BSA and treated with primary antibodies such as anti-phospho-NFκB p65 (#3033), anti-phospho-SAPK/JNK (#9251), anti-phospho-p38 (#9211), anti-phospho-p44/42 (#9101), anti-A20 (#5630) (Cell Signaling Technology, Danvers, MA, USA), anti-SOCS1 (sc-518028), and anti-β-actin HRP (sc-47778) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h at RT. After washing the membrane with TBST, HRP-conjugated anti-mouse (#31430, Invitrogen, Carlsbad, CA, USA) or anti-rabbit (sc-2357, Santa Cruz Biotechnology) secondary antibodies were incubated with membranes for 2 h at RT. After washing the membranes four times in TBST, protein bands were detected using ECL reagents (Thermo Fisher Scientific, Waltham, MA, USA) and exposed to X-ray film.

Choi K., Hong J., Kim K., Kim H., Lee S., Lee Y.J., & Chung D. (2022). Anti-Inflammatory Properties of MSF, a Lactiplantibacillus plantarum K8 Lysate Fermented with Filipendula glaberrima Extract. Applied Sciences, 12(5), 2602-2602.

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Immunoblotting of Apoptosis Regulators

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After stimulating THP-1 cells with aLTA for an appropriate time, protein samples were prepared using 2X reducing buffer. Proteins were separated using a 12% SDS-PAGE system (25 mM Tris, 250 mM glycine, 0.1% SDS) and transferred to a polyvinylidene fluoride (PVDF) membrane for overnight. The membrane was blocked with TBS-T buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, 5% skim milk) for 1 h at room temperature (RT). The primary antibodies and HRP-conjugated secondary antibodies diluted in TBS-T buffer were sequentially treated on the membrane, and the target protein was visualized using the enhanced chemiluminescence (ECL) reagents (GE Healthcare, UK) and X-ray film. Anti-Caspase-1, anti-Caspase-3, anti-Caspase-7, anti-Cytochrome C, anti-Bcl2, anti-A20, anti-Abin1, anti-SOCS-1 (Cell Signaling Technology, USA), and anti-β-actin (Santa Cruz Biotechnology, USA) antibodies were used in this study.

Jeon B., Kim H., & Chung D.K. (2022). Lipoteichoic Acid Isolated from Staphylococcus aureus Induced THP-1 Cell Apoptosis through an Autocrine Mechanism of Cytokines and SOCS-1-Mediated Bcl2 Inhibition. Microbiology and Biotechnology Letters, 50(2), 293-300.

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