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Nlrp3

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The NLRP3 is a laboratory equipment used for the detection and analysis of the NLRP3 protein. It is a key component of the NLRP3 inflammasome, which plays a crucial role in the innate immune response. The NLRP3 equipment provides researchers with a tool to study the NLRP3 protein and its involvement in various biological processes.

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Lab products found in correlation

Flag tag (flag), by Merck Group (3 mentions) Il 1β, by Cell Signaling Technology (2 mentions) Il 1β, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Merck Group (2 mentions) β actin, by Merck Group (2 mentions) Protein g sepharose bead, by GE Healthcare (1 mentions) Caspase 1, by Cell Signaling Technology (1 mentions) M per lysis buffer, by Thermo Fisher Scientific (1 mentions) P47phox, by Santa Cruz Biotechnology (1 mentions) Cox 2, by Santa Cruz Biotechnology (1 mentions) Actin hrp, by Merck Group (1 mentions) Erp72, by Cell Signaling Technology (1 mentions) Z vad fmk, by Merck Group (1 mentions) Gapdh hrp, by Proteintech (1 mentions) Tgn38, by Novus Biologicals (1 mentions) Histone h3, by Cell Signaling Technology (1 mentions) Lamp1, by Santa Cruz Biotechnology (1 mentions) Anti rabbit, by Thermo Fisher Scientific (1 mentions) Lc3a b, by Cell Signaling Technology (1 mentions) Bafilomycin a1, by Merck Group (1 mentions)

6 protocols using nlrp3

1

NLRP3 Inflammasome Activation Pathway

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Total protein from cell monolayers was harvested into M-PER lysis buffer (Thermo Fisher Scientific) and western blotting was performed to determine the expression of NLRP3 (Enzo life sciences), caspase-1, ASC, IL-1β (Cell Signaling Technologies) and IL-18 (MBL). Co-immunoprecipitation (Co-IP) was performed overnight at 4°C using NLRP3 antibody. Immunocomplexes were collected with Protein G Sepharose beads (GE Healthcare) for 1 h at 4°C prior to western blotting.
Ruiz P.A., Morón B., Becker H.M., Lang S., Atrott K., Spalinger M.R., Scharl M., Wojtal K.A., Fischbeck-Terhalle A., Frey-Wagner I., Hausmann M., Kraemer T, & Rogler G. (2016). Titanium dioxide nanoparticles exacerbate DSS-induced colitis: role of the NLRP3 inflammasome. Gut, 66(7), 1216-1224.
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2

Western Blot Analysis of Renal Proteins

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Cytoplasmic and nuclear proteins extracted from renal tissues as described above were used for Western blot analysis using antibodies against Nrf2, p47phox, COX-2, or IL-1β (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) or NLRP3 (Enzo Life Sciences Inc, Farmingdale, NY, USA). Antibodies against Lamin A (nuclear proteins) or β-actin (cytosolic proteins), both from Santa Cruz, were used as internal controls.
Ka S.M., Lin J.C., Lin T.J., Liu F.C., Chao L.K., Ho C.L., Yeh L.T., Sytwu H.K., Hua K.F, & Chen A. (2015). Citral alleviates an accelerated and severe lupus nephritis model by inhibiting the activation signal of NLRP3 inflammasome and enhancing Nrf2 activation. Arthritis Research & Therapy, 17, 331.
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3

Comprehensive Immunoblotting and Immunoprecipitation Procedures

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The following primary Abs were used for immunoblotting: NLRP3 (Cell Signaling 13158, 1:1000), IL-1β (Cell Signaling 12703, 1:1000), GFP (Cell Signaling 2955, 1:1000), TFEB (Cell Signaling 4240, 1:1000), CHOP (Cell Signaling 2895, 1:1000), LC3A/B (Cell Signaling clone D3U4C, 1:1000) Histone H3 (Cell Signaling clone D1H2, 1:2000), Myc (EMD Millipore clone 9E10, 1:2000), Flag (Sigma F1804, 1:1000), ASC (Santa Cruz sc-22514, 1:1000), LAMP1 (Santa Cruz clone H3A4, 1:2000), GAPDH-HRP (ProteinTech HRP-60004, 1:5000), and actin-HRP (Sigma A3854, 1:10,000). Anti-mouse (Cell Signaling 7076) or anti-rabbit (Cell Signaling 7074) HRP-conjugated secondary antibodies were used for ECL based detection. Primary Abs for immunofluorescence were NLRP3 (Enzo AlX-804-819), ASC (Santa Cruz sc-22514), ERp72 (Cell Signaling D70D12), and TGN38 (Novus, NBP1-03495). Alexa 568 conjugated polyclonal anti-mouse (Thermo Fisher, A11004) and anti-rabbit (Thermo Fisher, A11011) were used as secondary Abs prior to fluorescent imaging. For immunoprecipitation experiments, we used GFP antibodies coupled to magnetic beads (MBL, D153-9) and Flag antibodies coupled to agarose (Sigma, A2220) to pull down tagged proteins prior to immunoblotting. Cyclosporin A (R&D Systems, 1101) and Z-VAD-FMK (Sigma, V116) were used at 10 μM overnight, and Bafilomycin A1 (Sigma, B1793) at 100 nM for 4 h.
Shi C.S., Nabar N.R., Huang N.N, & Kehrl J.H. (2019). SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes. Cell Death Discovery, 5, 101.
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4

Detecting Inflammasome Proteins and Components

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For detecting caspase-1 and IL-1β in the supernatants and lysates, 20% vol RIPA buffer and 30% vol SDS-loading dye were added directly to the wells containing cells and media. The samples were boiled at 100°C for 15′–30′ and were run on 13% polyacrylamide gels. Cytosolic and nuclear fractions for protein detection were prepared by using Active Motif Nuclear Extract Kit. Co-immunoprecipitation assays were carried out with 5×106 cells (treated or untreated) in ice-cold RIPA buffer (without SDS and DOC) using Protein A Dynabeads (Novex/Life Technologies, Cat# 10001D) conjugated with specific antibodies, and immunoblotted for proteins of interest. Antibodies used were against Asc (Santa Cruz, Cat# sc-22514-R or Cell Signaling, Cat# 67824), IL-1β (R&D Systems, Cat# AF-401-NA), caspase-1 (Adipogen, Cat# AG-20B-0042), Aim2 (eBioscience, 14-6008), Nlrp3 (Enzo Life Sciences), Histone 3 (Abcam, Cat# ab1791), c/EBPβ (Santa Cruz, Cat# sc-150), Usf2 (Santa Cruz, Cat# sc-862), Gapdh (Sigma, Cat# G9295), β-actin (Sigma, Cat# A3854) HA-tag (Anti-HA-Peroxidase; Roche, Cat# 12 013 819 001), and FLAG-tag (Sigma, Cat# A8592). An affinity purified polyclonal antibody against p205 was generated using the following peptide: AGLDRLINFCERVPTL-amide) was generated (21st Century Biochemicals).
Ghosh S., Wallareth C., Covarrubias S., Hornung V., Carpenter S.B, & Fitzgerald K.A. (2017). The PYHIN protein p205 regulates the inflammasome by controlling Asc expression. Journal of immunology (Baltimore, Md. : 1950), 199(9), 3249-3260.
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5

Detecting NF-κB and NLRP3 Inflammasome Activation

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Protein extracts that were separated by SDS-PAGE and transferred onto PVDF membranes were probed with Abs against total NF-κB p65, phospho-NF-κB p65, caspase-1 p20, ASC (all from Cell signaling Technology, Danvers, MA), NLRP3 (Enzo life sciences, Farmingdale, NY), IL-1β (Santa Cruz Biotechnology, Santa Cruz, CA) and GAPDH (Santa Cruz Biotechnology). The probed membranes were washed and incubated with HRP-labeled secondary antibodies (Santa Cruz Biotechnology). The bands were visualized with the Pierce ECL Western blotting substrate (Thermo Scientific, Rockford, IL).
Shin M.S., Kang Y., Wahl E.R., Park H.J., Lazova R., Leng L., Mamula M., Krishnaswamy S., Bucala R, & Kang I. (2018). Macrophage migration inhibitory factor regulates U1-snRNP immune complex mediated activation of the NLRP3 inflammasome. Arthritis & rheumatology (Hoboken, N.J.), 71(1), 109-120.
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6

Western Blot Analysis of NLRP3 Inflammasome Activation

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BMDMs stimulated as per the experiment were lysed in buffer containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40 and 5 mM EDTA with fresh 1x Halt Protease inhibitor cocktail (Promega #1861279). Homogenized lysates were resolved on 14% SDS-PAGE, and transferred to 0.2 μM PVDF membrane. Membranes were blocked with 5% non-fat dry milk (w/v) and probed with antibodies diluted in 1X PBS and 0.05% Tween-20 (v/v). The antibodies used were pro-IL1β (R&D Systems, AF-401-NA), caspase-1 (Santa Cruz Biotechnology, sc-514), gasdermin D (Abcam, AB209845), β-actin (Sigma), Nlrp3 (Enzo Life Sciences, clone cryo-2), Gapdh (Sigma, G9295), Flag (Sigma, A8592), KDEL (Enzo Life Sciences 10C3), Tom20 (Abcam, AB186734), VDAC (Cell Signaling, 4866S), HSP60 (Santa Cruz, 13115). The Mm47 antibody was custom made by Thermo Fisher against the immunogenic residue 22–47 of Mm47. Membranes were probed with horseradish peroxidase-conjugated anti-mouse (Bio-Rad, 172–1011) and anti-rabbit (Bio-Rad, 170–6515) or anti-Goat (Bio-rad, 172–1034) and developed using ECL chemiluminescent substrate (Pierce).
Bhatta A., Atianand M., Jiang Z., Crabtree J., Blin J, & Fitzgerald K.A. (2019). A mitochondrial micropeptide is required for activation of the Nlrp3 Inflammasome. Journal of immunology (Baltimore, Md. : 1950), 204(2), 428-437.
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