PBMCs cellular lysates were prepared by gentle shaking and applying 10 pulses of sonication (Branson Sonifier 150) in RIPA buffer supplemented with protease inhibitor PhenylMethylSulfonylFluoride 1 mM. Similarly, plasma samples were diluted in RIPA buffer (1:1, v/v) and sonicated as previously described. Protein content was determined using the Bradford method and electrophoresis was carried out in 12% acrylamide SDS–PAGE, loading 60 µg samples. Then, proteins were transferred to PVDF membranes (Biorad, Hercules, CA, USA) and incubated with primary antibody solutions [1:1000] for NLRP3 (D4D8T, Cell Signaling), IL-1β (D2F3B, Cell Signaling), caspase-1 (2225, Cell Signaling), and gasdermin-D (sc-376318, Santa Cruz) quantification. Membranes were then incubated with their respective secondary antibody [1:2000] and immunolabeled proteins were detected by chemiluminescence method using Western Bright Sirius HRP substrate (Advansta, San Jose, CA, USA). Western blot images were finally quantified using Image-Lab software (Biorad, Hercules, CA, USA).
D4d8t
Manufactured by Cell Signaling Technology
Sourced in United States, China
D4D8T is a cell culture dish product designed for use in cell-based experiments. It features a polystyrene surface that has been treated to promote cell attachment and growth. The dish provides a controlled environment for culturing cells.
Automatically generated - may contain errors
Lab products found in correlation
Ag 20b 0042, by Adipogen (2 mentions)
Westernbright sirius hrp substrate, by Advansta (1 mentions)
Sonifier 150, by Emerson (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti ha antibody c29f4, by Cell Signaling Technology (1 mentions)
Anti flag m2 bead, by Merck Group (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ab219800, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab48394, by Abcam (1 mentions)
Ap0705, by ABclonal (1 mentions)
A19566, by ABclonal (1 mentions)
Gapdh, by Merck Group (1 mentions)
Nigericin, by Merck Group (1 mentions)
Dansylsarcosine piperidinium salt, by Merck Group (1 mentions)
Antibodies against, by Cell Signaling Technology (1 mentions)
Anti mouse igg dylight 488, by Abbkine (1 mentions)
Human il 1β elisa kit, by BD (1 mentions)
9 protocols using d4d8t
1
NLRP3 Inflammasome Western Blot Protocol
2
Immunoblot Analysis of NLRP3 Inflammasome
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Equal amounts of supernatants or cell lysates were loaded to SDS-PAGE, and immunoblot was performed by anti-cleaved IL-1β (Asp117) Ab (Cell Signaling Technology Inc., Danvers, MA), anti-Caspase-1 (p20) Ab (Casper-1, AdipoGen, San Diego, CA) or anti-NLRP3 Ab (D4D8T, Cell Signaling Technology Inc.).
3
Immunoblotting and Immunoprecipitation Protocols
4
Western Blot Analysis of Pancreatic Proteins
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Briefly, pancreatic tissues were lysed in RIPA lysis buffer, and total proteins were extracted. Concentrations of proteins were measured using the BCA protein assay kit (Pierce, Thermo Fisher Scientific) following the commercial instructions. Equivalent protein (approximately 30 μg/sample) was separated by SDS-PAGE gel and then transferred to PVDF membranes. Then the membranes were blocked for 1 hour in 5% nonfat milk. Afterward, membranes were incubated with primary antibodies against GAPDH (1:1,000 dilution; Sigma-Aldrich), NLRP3 (1:1,000 dilution; D4D8T, Cell Signaling Technology), GSDMD (1:500 dilution; ab219800, Abcam), caspase-1 (1:500 dilution; AG-20B-0042, AdipoGen), STAT3 (1:1,000 dilution; A19566, Abclonal), phosphorylated STAT3 (1:1,000 dilution; AP0705, Abclonal), Beclin 1 (1:1,000 dilution; A21191, Abclonal), LC3B (1:1,000 dilution; ab48394, Abcam), Bax (1:1,000 dilution; 29057, Sabbiotech), and Bcl-2 (1:1,000 dilution; ab196495, Abcam) overnight at 4°C. Finally, HRP-conjugated secondary antibodies were applied for 1 hour. The proteins were detected and analyzed by an ECL Plus chemiluminescence imaging system (Tanon).
5
Inflammasome Activation and Cytokine Release
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Phorbol-12-myristate-13-acetate (PMA), LPS, nigericin, ATP, and dansylsarcosine piperidinium salt (DSS) were purchased from Sigma-Aldrich. Caspase-1 inhibitor (VX-765) was purchased from Selleck. Recombinant human IL-1β protein (200-01B) and human IL-1RA (200-01RA) were obtained from Peprotech. Recombinant mouse IL-1β protein (401-ML/CF) and recombinant mouse IL-1RA (480-RM/CF) were purchased from R&D Systems. Trizol reagent was from Invitrogen, and Lipofectamine 2000 was from Invitrogen. The human IL-1β ELISA kit was purchased from BD Biosciences, the mouse IL-1β and tumor necrosis factor (TNF)-α ELISA kits were purchased from R&D Systems, and the LPS ELISA kit was purchased from Expandbio (Beijing, China).
Antibodies against NLRP3 (D4d8T), caspase-1 (D7F10), and IL-1β (D3U3E and 3A6) were purchased from Cell Signaling Technology. Antibody against DENV-NS3 (GTX124252) and DENV-Prm (GTX128092) were purchased from Genetex. Anti-β-actin antibody (66009) was purchased from Proteintech. Antibody against ASC (sc-271054) was purchased from Santa Cruz Biotechnology. Anti-mouse IgG Dylight 649, anti-mouse IgG Dylight 488, anti-rabbit IgG Dylight 649, and anti-rabbit IgG fluorescein isothiocyanate (FITC) were purchased from Abbkine.
Antibodies against NLRP3 (D4d8T), caspase-1 (D7F10), and IL-1β (D3U3E and 3A6) were purchased from Cell Signaling Technology. Antibody against DENV-NS3 (GTX124252) and DENV-Prm (GTX128092) were purchased from Genetex. Anti-β-actin antibody (66009) was purchased from Proteintech. Antibody against ASC (sc-271054) was purchased from Santa Cruz Biotechnology. Anti-mouse IgG Dylight 649, anti-mouse IgG Dylight 488, anti-rabbit IgG Dylight 649, and anti-rabbit IgG fluorescein isothiocyanate (FITC) were purchased from Abbkine.
6
Western Blot Analysis of Inflammasome Proteins
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Cells were lysed in KalB lysis buffer (50 mM Tris, 150 mM NaCl, 1% Trition X-100 and 1 mM EDTA, 1% NaF, 1% NaVO4, 1 mM PMSF, and protease inhibitor cocktail, pH 7.4). Proteins from cell supernatants were extracted by methanol–chloroform precipitation. Protein samples were resolved on 15% NuPAGE® Bis-Tris gels (Novex, Thermo Fisher Scientific) and then transferred onto PVDF membrane. Membranes were blocked in 5% (w/v) milk powder in Tris-buffered saline with Tween-20 (TBS-T) (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% (v/v) Tween-20) for 1 h at room temperature. Membranes were incubated with primary antibodies diluted in 5% (w/v) milk powder in TBS-T overnight at 4 °C and then with horseradish peroxidise-conjugated secondary antibody diluted in 5% (w/v) milk powder in TBS-T for 1 h at room temperature. Membranes were developed with Amersham ECL Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) and images either taken on film or using a Fujifilm LAS-4000 luminescent reader. Primary antibodies used were mouse anti-caspase-1 (AG-20B-0042, Adipogen), rabbit anti-IL-1β (ab9722, Abcam), rabbit anti-NLRP3 (D4D8T, Cell Signaling Technology), rabbit anti-MIF (ab7207, Abcam), rabbit anti-ASC (sc-22514-R, Santa Cruz), and mouse anti-vimentin (sc-373717, Santa Cruz).
7
Western Blot Analysis of Hepatic Proteins
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Total hepatic proteins were extracted using lysis buffer (Beyotime) containing a mixture of phosphatase and protease inhibitors (MedChemExpress, Monmouth Junction, USA). Equal amounts of the protein samples were separated by 8%–15% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Immobilon-P; Millipore, Billerica, USA). After incubation with 5% skimmed milk, the membranes were incubated with primary antibodies against IL-1β (SC-52012; Santa Cruz), tumor necrosis factor (TNF)-α (SC-52746; Santa Cruz), Caspase-1 (SC-56036; Santa Cruz), cholesterol 7α-hydroxylase (CYP7A1, SC-518007; Santa Cruz), sterol 12α-hydroxylase (CYP8B1, SC-101387; Santa Cruz), β-actin (SC-47778; Santa Cruz), Gasdermin D (GSDMD, AF4012; Affinity Biosciences, Cincinnati, USA), FXR (25055-1-AP; Proteintech, Chicago, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, FD0063; Fude Biological Technology, Hangzhou, China), NLRP3 (D4D8T; Cell Signaling Technology), and ASC (SC-514414; Santa Cruz) at 4°C for 12 h. The membranes were then incubated with peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG antibodies (Fude Biological Technology). The bands on membranes were visualized using an enhanced chemiluminescence kit (Fude Biological Technology) and detected using the Quick Chemi 5200 chemiluminescence imaging system (Monad Biotech, Suzhou, China).
8
Quantitative analysis of NLRP3 protein
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Total protein was extracted from FGR (n = 5) and gestation-matched controls (n = 5). Immunoblotting for NLRP3 was performed as previously described [36 (link)]. Rabbit monoclonal anti-human NLRP3 (D4D8T, #15101, 0.1 µg/mL, 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA) was used as the primary antibody, and the immunoreactive protein was visualised using a peroxidase-conjugated donkey anti-rabbit IgG-HRP secondary antibody (Zymed, Waltham, MA, USA), and detected by an enhanced chemiluminescence system (Bio-Rad, Hercules, CA, USA). The level of immunoreactive proteins was determined semi-quantitatively using scanning densitometry (Image Quant, Molecular Dynamics Inc., Sunnyvale, CA, USA), relative to rabbit polyclonal GAPDH (1 μg/mL, 1:1000 dilution, Imgenex Technology Corporation, British Columbia, Canada) [36 (link)].
9
Immunoprecipitation of NLRP3 for Protein Analysis
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Cells were washed with PBS and lysed in RIPA buffer or Co-IP lysis buffer (50 mM Tris pH 7.8, 50 mM NaCl, 0.1% NP-40, 10 % Glycerol, cOmplete protease inhibitor and PhosSTOP (Roche), PMSF (0.2 mM). Nuclei were removed by centrifugation (10 min, 1000×g) and protein concentration was normalized after measurement by bichinonic acid assay (BCA; Thermo Fisher Scientific).
Endogenous NLRP3 was immunprecipitated using an NLRP3 antibody (D4D8T, Cell signaling) bound to protein G Dynabeads (Life Technologies). IP was performed for 4 h at 4 °C. Samples were denatured with LDS buffer supplemented with reducing agent (Thermo Fisher) for 10 min at 85 °C.
Co-immunprecipitation of NLRP3-mCitrine was perfomed in GFP-trap plates (Chromotec) for 2.5 h at 4 °C shaking or using a tRFP antibody (AB233, evrogen) coupled to protein G Dynabeads. Samples were denatured as described before.
Endogenous NLRP3 was immunprecipitated using an NLRP3 antibody (D4D8T, Cell signaling) bound to protein G Dynabeads (Life Technologies). IP was performed for 4 h at 4 °C. Samples were denatured with LDS buffer supplemented with reducing agent (Thermo Fisher) for 10 min at 85 °C.
Co-immunprecipitation of NLRP3-mCitrine was perfomed in GFP-trap plates (Chromotec) for 2.5 h at 4 °C shaking or using a tRFP antibody (AB233, evrogen) coupled to protein G Dynabeads. Samples were denatured as described before.
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