Punicalagin (purity = 98%, CAS: 65995-63-3) was purchased from Herbpurify Co., Ltd. (Chengdu, China). A urea assay kit, a creatinine assay kit (sarcosine oxidase) and a urine microalbumin assay kit were purchased from the Nanjing Jiancheng Bioengineering Institute (China). A mitochondrial membrane potential assay kit with JC-1 and a tissue mitochondria isolation kit were purchased from Beyotime Biotechnology (Shanghai, China). Antibodies targeting the following proteins were used in this study: NLRP3 (A12694, Abclonal), Trx (A01219-1, BOSTER), TXNIP (sc-166234, Santa Cruz; A9342, Abclonal), IL-1β (A1112, Abclonal), caspase-1 (BM4291, BOSTER), GSDMD (A10164, Abclonal), β-actin (60008-1-Ig, Proteintech), NOX4 (14347-1-AP, Proteintech).
Gsdmd
Manufactured by ABclonal
Sourced in China
GSDMD is a lab equipment product offered by ABclonal. It is a protein that plays a key role in the process of pyroptosis, a form of programmed cell death. GSDMD 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
Nlrp3, by ABclonal (5 mentions)
Il 1β, by ABclonal (4 mentions)
Caspase 1, by ABclonal (4 mentions)
β actin, by ABclonal (2 mentions)
Bcl2 associated x (bax), by ABclonal (2 mentions)
Bcl 2, by ABclonal (2 mentions)
Caspase 9, by ABclonal (2 mentions)
Caspase 3, by ABclonal (2 mentions)
Phosphorylated p 65, by ABclonal (2 mentions)
Nox4 14347 1 ap, by Proteintech (1 mentions)
Txnip, by Santa Cruz Biotechnology (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Tissue mitochondria isolation kit, by Beyotime (1 mentions)
β actin 60008 1 ig, by Proteintech (1 mentions)
Urea assay kit, by Nanjing Jiancheng (1 mentions)
Confocal laser scanning microscopy, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Biosharp (1 mentions)
Claudin 1, by Affinity Biosciences (1 mentions)
Nf κb p65, by Abmart (1 mentions)
P iκb, by Abmart (1 mentions)
14 protocols using gsdmd
1
Punicalagin Modulates Oxidative Stress
2
Quantifying Pyroptosis in Stomach Muscle
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In order to detect the level of pyroptosis in muscular stomach tissue, NLRP3 and GSDMD immunofluorescence double staining was detected. According wax blocks and sections in 2.2., the details of IF staining method was referred to Shi's article (Shi et al., 2023 (link)). Nuclei were stained by DAPI (Biosharp, China). Primary antibodies NLRP3 (Abclonal, China) and GSDMD (Abclonal, China) were both diluted 1:200. Low and high magnification images were acquired by a laser scanning confocal microscopy (Leica, Germany). Image J softwawre was used to quantify the relative fluorescence intensity of NLRP3+ and GSDMD+ in muscular stomach tissue.
3
Immunofluorescence Analysis of NLRP3, GSDMD, and ZO-1 in RIMVECs
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The sterilized coverslips were placed into the wells of a 24-well plate, and then RIMVECs were added to each plate at the concentration of 1 × 105 cells/well in 600 μL DMEM. When the cells reached 90% confluence onto the coverslip, they were divided into the same groups as described in the in vitro experiment paragraph. At the end of the incubation time, the cells were fixed with 500 μL paraformaldehyde (PFA) for 40 min, followed by permeabilization with 0.2–0.5% Triton X-100 for 20 min. The cells were washed three times with phosphate buffered saline (PBS) before and after each step and then incubated with the following primary antibodies at 4°C overnight: NLRP3 (1:1000, ABclonal), GSDMD (1:1000, ABclonal), caspase-1 (1:1000, ABclonal), and ZO-1 (1:1000, Affinity Biosciences). Next, the secondary antibody (1:5000 dilution in 5% BSA, ABclonal) was added, and the cells were incubated at room temperature for 1 h in the dark. One μg/mL 4’,6-diamidino-2-phenylindole (DAPI) was added to each well, and the cells were incubated at room temperature for 20 min in the dark. After washing, the coverslips were removed from the wells and blotted to remove any excess water. Each coverslip was placed onto a microscope slide, and the slides were sealed with nail polish and observed under a confocal fluorescence microscope (NIKON Ti-S, Tokyo, Japan).
4
Western Blot Analysis of Inflammasome Signaling
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RIMVECs were seeded, divided into groups, and treated as described in the in vitro experiment paragraph. Total proteins were extracted using the radio immunoprecipitation assay (RIPA) cell lysis buffer for 10 min, and protein concentration was determined using the bicinchoninic acid (BCA) protein detection kit (Beyotime Biotechnology, China). A standard western blot protocol was performed, and the following primary antibodies were used overnight at 4°C: TLR4 (1:800) (ABclonal, Wuhan, China), NLRP3 (1:1000) (ABclonal), GSDMD (1:1000) (ABclonal), caspase-1 (1:1000) (ABclonal), ASC (1:1000) (ABclonal), NF-κB p65 (1:1000) (Abmart, Shanghai, China), p-NF-κB p65 (1:400) (Abmart), p-I-κB (1:1000) (Abmart), I-κB (1:1000) (Abmart), IL-18 (1:1000) (Affinity Biosciences, Colorado, USA), IL-1β (1:1000) (Affinity Biosciences), claudin-1 (1:1000) (Affinity Biosciences), claudin-2 (1:1000) (Affinity Biosciences), ZO-1 (1:1000) (Affinity Biosciences), and β-tubulin (1:1000) (Affinity Biosciences) used as loading control, followed by the incubation with HRP (Horseradish Peroxidase)-conjugated secondary antibodies (1:5000) (ABclonal) for 1 h at room temperature. The blots were visualized using a Tanon 5200 chemiluminescence imaging system (Shanghai, China) and quantified using the ImageJ software (National Institutes of Mental Health, Bethesda, MD, USA).
5
Protein Extraction and Western Blot Analysis
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Cells were lysed using the PRO-PREPTM Protein Extraction solution (iNtRon Biotech, Seongnam-Si, Republic of Korea). Total proteins were transferred to a polyvinylidene difluoride membrane and blocked with 5% skimmed milk for 1 h. Specific proteins were detected with primary antibodies: NLRP3 (A12694), GSDMD (A20197), IL-1β (A1112), caspase-3 (A19654), caspase-9 (A11451), BNIP3 (A5683), Cyt-c (A1561), Bax (A19684), Bcl-2 (A19693), β-actin (AC026), p-mTOR (S2448) (AP0115), mTOR (A2445), p-PI3K (AP0845), PI3K (A4992), p-Akt (AP0637), Akt (A17909), P62 (A19700), and LC3B (A19665), which were all bought from ABclonal Technology Co. Ltd., Wuhan, China. The blots were then treated with horseradish peroxidase-conjugated secondary antibodies. Bands were visualized using the ECL reagent and FluorChem M system (ProteinSimple, SFO, San Jose, CA, USA)
6
Antibody-mediated Regulation of NLRP3 and NF-κB Signaling
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Antibodies targeting the following proteins were purchased from ABclonal Biotech Co., Ltd. (USA): HIF-1α (#A11945), HIF-1α (#A1544), NLRP3 (#A6345), NLRP12 (#A6671), NLRC4 (#A7382), CASP1 (#A0964), CASP8 (#A0215), IL-1β (#A1112) and GSDMD (#A10164). Anti-cleaved-CASP8 (#8592S) and anti-phospho-NF-kB P65 (#3033S) antibodies were obtained from Cell Signaling Technology. An IL-1β neutralizing antibody (#14–7012-85) was obtained from eBioscience. The CASP1 inhibitor (CASP1 inh) Z-YVAD-FMK (YVAD, #ab141388) was obtained from Abcam. The NF-kB P65 inhibitor JSH-23 (#S7351) was purchased from Selleckchem. Anti-β-actin (#AP0060) and secondary antibodies were purchased from Bioworld Technology. Lipofectamine 3000 was purchased from Invitrogen.
7
Ferroptosis and Apoptosis Pathway
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The MLKL, FDX1, GSDMD, p-MLKL, RIP1, p-RIP1, GS, GLS, ACOT2, BAAT, GPx4, ACSL4, LC3, TH and GAPDH primary antibodies were purchased from ABclonal Technology (Wuhan, China). The SMS, HSP70, caspase3 and capase8 primary antibody were purchased from ZENBIO Technology (Chen du, China). The capase1 primary antibody was purchased from ThermoScientific Technology (Massachusetts, United States). The beclin-1 primary antibody was purchased from Cell Signaling Technology (Boston, United States). The details of antibodies were shown in Table S4 . IL-1ß, TNF-α and IL-10 commercial reagent kits were purchased from Yi Fei Xue Biotechnology (Nanjing, China). Lipid Peroxidation MDA Assay Kit, Reduced glutathione/Oxidized glutathione (GSH/GSSG) Assay Kit and NADP+/NADPH Assay Kit with WST-8 were purchased from Beyo-time Biotechnology (Shanghai, China).
8
Investigating EEBR's Anti-Cancer Effects
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The A549 and H1299 human lung cancer cells were provided by the Pharmacology Laboratory of Harbin Medical University, and the most recent STR identification was in September 2023 (Figure S1A,B ). BALB/c Nude mice were purchased from Beijing Viton Lihua Laboratory Animal Technology Co. The experimental compound EEBR was provided by the Laboratory of Natural Drugs and Medicinal Chemistry, Harbin Medical University, while cisplatin was obtained from Qilu Pharmaceutical Co., LTD. VX‐765 and BAY 11‐7082 inhibitors were purchased from Dalian Meilun Biotechnology Co., LTD. Trypsin, 1640 medium and penicillin/streptomycin were supplied by Gibco (Carlsbad, CA, USA), while fetal bovine serum (FBS) was obtained from Clark Corporation, USA. The Cell Counting Kit‐8 (CCK8) was purchased from Dojindo and the BCA protein quantitation kit from Beyotime. The antibodies specific for NF‐κB (cat# A10609), NLRP3 (cat# A5652), cleaved Caspase‐1 (cat# A23429) and GSDMD (cat# A20728) were purchased from ABclonal; N‐terminal GSDMD (cat# ab215203) was purchased from Abcam; β‐actin (cat# bs‐0061R), Caspase‐1 (cat# bs‐10442R), interleukin‐18 (IL‐18) (cat# bs‐4986R), interleukin‐1 beta (IL‐1β) (cat# bs‐25615R) antibodies were obtained from Bioss.
9
TXNIP and NLRP3 Inflammasome Pathway
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Protein from pancreatic tissue and INS-1 cells lysates were collected. Western blot procedures were processed as standard protocol with specific antibodies against TXNIP (1:1,000, Abcam, UK), GSDMD (1:1,000, Abclonal, USA), caspase-1 (1:1,000, Abclonal, USA), NLRP3 (1:1,000, Adipogen, USA), and β-actin (1:5,000, Sigma, USA). The blots were visualized by ChemiDoc™ Touch Imaging System (Bio-Rad, CA, USA). Western blot image was analyzed and quantified by using Image J software (NIH, Bethesda, MD, USA). Relative expression of the protein was normalized with β-actin expression.
10
Renal Cell Culture and Urate Metabolism
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Cell culture medium and human primary renal PTECs were provided by ScienCell (San Diego, CA, United States). Anti-SLC2A9 (URAT1) antibodies, TSG101, CD63, caspase-1, GSDMD, IL-1β, caspase-3, caspase-8, caspase-9, cytochrome c, Bcl-2, and Bax were purchased from ABclonal (Wuhan, China). Oxonic acid (OA) and UA were provided by Sigma (St. Louis, MO, United States). CIAS1/NALP3, GAPDH, and anti-GLUT9 were provided by Abcam (Cambridge, United Kingdom). In addition, the CCK-8 assay kit was provided by Jiwei Biological Technology (Shanghai, China).
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