NK92 cells or U937 cells were lysed using Laemmli lysis buffer (Sigma-Aldrich). Blots were prepared and then blocked with 5% dry milk solution in TBST for 1 h. Primary antibodies to HCA1, HAC2, HCA3, gasdermin-D, DNMT3A, DNMT3B or Caspase-1 (Abcam, Cambridge, UK) were used. HRP conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Cell Signaling Technology, Danvers, MA, USA), were diluted in fresh 5% dry milk in TBST solution and incubated with the blots for 1 h at room temperature. HRP was detected using BioRad ECL Western blotting detection reagent (BioRad, Hercules, CA, USA). Primary antibody for Actin (Cell Signaling Technology) was used to confirm loading equality.
Gasdermin d
Manufactured by Abcam
Sourced in United Kingdom, United States
Gasdermin D is a protein that plays a key role in the process of programmed cell death known as pyroptosis. It is involved in the formation of pores in the cell membrane, leading to cell lysis and the release of inflammatory contents.
Automatically generated - may contain errors
Lab products found in correlation
Caspase 1, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Nlrp3, by Abcam (3 mentions)
Il 18, by Abcam (3 mentions)
Caspase 11, by Abcam (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Il 1β, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gasdermin e, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Laemmli lysis buffer, by Merck Group (1 mentions)
Dnmt3a, by Abcam (1 mentions)
Dnmt3b, by Abcam (1 mentions)
Ecl western blot detection reagents, by Thermo Fisher Scientific (1 mentions)
Il 18, by Proteintech (1 mentions)
Anti rabbit or anti mouse secondary antibodies, by Jackson ImmunoResearch (1 mentions)
β actin, by GeneTex (1 mentions)
17 protocols using gasdermin d
1
Western Blot Analysis of Apoptosis-Related Proteins
2
Western Blot Analysis of Inflammasome Proteins
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Mouse kidney tissue or MTECs were lysed in RIPA buffer with PMSF and phosphatase inhibitors (RIPA:PMSF:phosphatase inhibitors = 100:1:1). The protein concentrations were determined using a BCA Protein Assay Kit (Beyotime, China). The protein samples were then transferred to PVDF membranes (Millipore, Sigma). The following primary antibodies were used: Gasdermin D (Abcam, UK), caspase-1 (Abcam, UK), caspase-11 (Abcam, UK), IL-1β (CST, USA), IL-18 (Proteintech, USA), TLR4 (Proteintech, USA), NLRP3 (Abcam, UK), ASC (Abcam, UK), and β-actin (GeneTex, USA). Anti-mouse or anti-rabbit secondary antibodies (Jackson ImmunoResearch, USA) were used to bind the primary antibodies at room temperature for 1 h. Signals were developed with ECL Western blot detection reagents (Thermo Fisher Scientific, USA). Finally, quantification was performed using ImageJ v1.8.0 software (National Institutes of Health, Bethesda, MD, USA).
3
Western Blot Analysis of NLRP3 Inflammasome Pathway
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Total proteins extraction process was performed by using the RIPA lysis buffer purchased from Beyotime (Shanghai, China), and the total proteins were separated according to their molecular weight by using the 10% SDS-PAGE, which were then transferred onto the PVDF membranes (Millipore, MA, USA) and were blocked with the 5% nonfat milk. Then, the above membranes were probed with the primary antibodies against NLRP3 (1:1500, Abcam, UK), Gasdermin D (1:1500, Abcam, UK), cleaved caspase-1 (1:1500, Abcam, UK), IL-1β (1:2000, Abcam, UK), IL-18 (1:1000, Abcam, UK) and GAPDH. (1:2500, Abcam, UK) at 4 °C overnight, which were subsequently incubated with the secondary antibodies (1:3000, Abcam, UK) for 2 h at room temperature. An Enhanced Chemiluminescence (ECL, Bio-Rad, CA, USA) was used to visualize the protein bands, and the gray values were measured by the Image J software to represent protein expressions.
4
Antibody Profiling for Flow Cytometry and Western Blotting
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The following antibodies were used for flow cytometry: CD11b-PeCy7 (clone M1/70, eBioscience), F4/80 (clone BM8, eBioscience), and fixable viability dye (eBioscience). The following antibodies were used for western blotting: phospho-p65, NF-κB2, phospho-ERK, phospho-JNK, phospho-p38, total ERK, total JNK, and total p38 from Cell Signaling Technologies. Total p65 was purchased from Santa Cruz. TRAF2, gasdermin D and caspase-11 were purchased from Abcam, caspase-1 from Adipogen and IL-1β from RnD. cIAP1 was purchased from Human Atlas. RIPK1 and XIAP was purchased from BD Biosciences. Secondary antibodies for western blotting such as donkey anti mouse/rabbit/rat IgG conjugated to HRP are from SouthernBiotec, the donkey anti goat IgG was purchased from Santa Cruz. The neutralizing antibody against TNF (at 200 ng/mL, MP6-XT22) was purchased from BioLegend.
5
Immunoblotting of Apoptosis Regulators
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Proteins were extracted using Lysis Buffer (Tris-HCl 50 mM, NaCl 150 mM, EDTA 5 mM and Triton-X100 1%) and Cocktail Protease Inhibidor (04693159001, Roche). 14% polyacrilamide gelswere transferred to PVDF membrane using a semi-dry system. The membrane was blocked for 1 hour and incubated overnight at 4 °C with the primary antibody (Anti-pro-caspase-1, Gasdermin D, pro-IL-1β and ASC; Abcam). The membrane was then incubated for 1 hour with secondary antibody (Jacksons Immuno Research). The anti-β-actin antibody (Aldrich) was used as loading control. The bands were revealed using Chemiluminescent Substrate (Westar Supernova XLS3L and XLS3P) by Image Quant LAS 4000 (GE Healthcare Life Sciences). The bands were analyzed with the ImageJ software (Version 1.8) for densitometry analysis.
6
Caspase and Gasdermin Protein Analysis
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MuSC were lysed in phenylmethanesulfonyl fluoride (PMSF), and protein content was quantified with bicinchoninic acid, loaded onto SDS-PAGE gels, and transferred onto nitrocellulose membranes as described previously [12 (link)]. Immunoblots were probed with antibodies to caspase-1 (Cell Signaling, Danvers, MA, USA, #2225S), caspase-5 (Cell Signaling, #46680S), caspase-3, gasdermin D (Abcam, #210070), and actin (Cell Signaling, #3700T). Chemiluminescence was used to detect protein expression. ImageJ analysis program [17 (link)] was for semi-quantitative analysis of each protein normalized to actin.
7
Investigating Inflammasome Activation and Apoptosis
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LPS, ATP, nigericin, poly dA:dT, staurosporine, cytochalasin D, valinomycin, and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, USA). JC-1 and MitoSox were obtained from Invitrogen (San Diego, CA, USA). zVAD-FMK,and ac-YVAD-CMK were purchased from Bachem (Torrance, CA, USA). Mammalian expression constructs for mouse Sarm1 (pGW1-Myc-Sarm1) was purchased from Addgene (Watertown, MA, USA). The following antibodies were used for detecting mouse caspase-1 (Adipogen, San Diego, CA, USA), NLRP3 (Adipogen), IL-1β (R&D Systems, Minneapolis, MN, USA), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (Santa Cruz Biotechnology), caspase-3 (Cell Signaling, Beverly, MA, USA), gasdermin D (Abcam, Cambridge, MA, USA), and Sarm1 (Cell Signaling).
8
Glioma Cell Line Characterization
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Normal human brain cells and glioma cell lines (U251, SHG-44, U87) cells were purchased from Shanghai CAS cell bank; HIF-1α overexpressing adenovirus expression vector AAVrh.10 HIF-1α with adenovirus capsid AAVrh.10 Null was purchased from Jinan Sike Biotechnology Co. Apoptosis-associated speck-like protein containing caspase-recruitment domain (ASC), caspase-1, gasdermin-D (GSDMD), gasdermin-E (GSDME), and transforming growth factor β1 (TGF-β1) antibodies were purchased from Abcam. MTT, Trizol, and de-RNAase treated items were purchased from Sigma; RT-PCR primers were synthesized by Suzhou Jin Wei Zhi Biotechnology Co. DMEM medium, fetal bovine serum, trypsin, and other cell culture reagents were purchased from Gibco, USA, and the BCA protein kit was purchased from Biyuntian Technology Co.
9
Immunoblotting for Inflammasome Proteins
10
Quantification of Cytokine Secretion
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Human cytokines secretion were quantified by ELISA kits, according to the manufacturer’s instructions, IL-1B (Thermo Fisher Scientific, (88-7261-77), IL-6 (BD, 555220), IL-16 (R&D, DY316), Caspase 3 (Invitrogen, BMS2012INST), Gasdermin E (AG-45B-0024-KI01), Gasdermin D (Abcam, ab272463), Cleaved Caspase-3caspase-3 (Abcam, ab220655). Before use, the samples were diluted 1/3 to 1/5 with their respective assay diluents. IL-18 was quantified using IL-18 Reporter HEK 293 Cells according to the manufacturer’s instructions (InvivoGen). HMGB1 was quantified by ELISA according to the manufacturer’s instructions (Novus Biologicals).
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