Rabbit anti gsdmd

Manufactured by Abcam

Sourced in United Kingdom

Rabbit anti-GSDMD is a primary antibody that specifically recognizes the Gasdermin D (GSDMD) protein. GSDMD is a key executor of pyroptosis, a form of programmed cell death. This antibody can be used to detect and study the GSDMD protein in various experimental settings.

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Close spelling variants of this product

GSDMD (79 citations) Anti-GSDMD (23 citations) Rabbit anti- (5 citations) Rabbit anti-mouse GSDMD (EPR20859, (2 citations)

5 protocols using rabbit anti gsdmd

Western Blot Analysis of Neonatal Rat Brain

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Neonatal rats were deeply anesthetized with anhydrous 2-butanol and then sacrificed. Cortical tissues of the neonatal rat brain and HAPI microglial cells were lysed into homogenate in lysis buffer, and the extracted proteins (20 μg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Millipore, Burlington, MA, USA). Membranes were incubated with mouse anti-TIGAR (1:1000; Cat# sc-166290; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Caspase-1 (1:1000; Cat# ab207802; Abcam, Cambridge, UK), rabbit anti-GSDMD (1:1000; Cat# ab219800; Abcam), anti-GSDMD-N (1:1000; Cat# ab215203; Abcam), rabbit anti-IL-1β (1:1000; Cat# ab9787; Abcam), or mouse anti-β-actin (1:10 000; Cat# ab008; MULTI SCIENCES, Hangzhou, China) overnight at 4°C. The membranes were then incubated with peroxidase-conjugated rabbit/mouse secondary antibodies (1:5000; Cat# 111-035-003/115-035-003; Jackson, Lancaster, PA, USA), and bands were detected with an AmerSham Imager 600 (GE, Boston, MA, USA) and analyzed using ImageJ analysis software (National Institutes of Health, Bethesda, MD, USA).

Tan L.L., Jiang X.L., Xu L.X., Li G., Feng C.X., Ding X., Sun B., Qin Z.H., Zhang Z.B., Feng X, & Li M. (2020). TP53-induced glycolysis and apoptosis regulator alleviates hypoxia/ischemia-induced microglial pyroptosis and ischemic brain damage. Neural Regeneration Research, 16(6), 1037-1043.

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Inflammasome and α-Synuclein Regulation

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Dulbecco's Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS) and other cell culture reagents, goat anti-mouse- AlexaFluor594 (H + L) antibody, goat anti-rabbit-AlexaFluor488 (H + L) antibody and rabbit IgG isotype control antibody were obtained from Thermo Fisher Scientific (Invitrogen). si-m-PYCARD/ASC RNAs were obtained from Guangzhou RiboBio Co., Ltd. Mouse anti-NLRP3 (Cat# AG-20B-0014), mouse anti-caspase-1 (Cat# AG-20B-0042) and rabbit anti-ASC (Cat# AG-25B-0006) were purchased from AdipoGen Life Sciences. Rabbit anti-α-synuclein (Cat# 4179), rabbit anti-Phospho-α-synuclein (Cat# 23706), rabbit anti-phospho-NF-κB p65 (Cat# 3033) and mouse anti-IL-1β (Cat# 12242) were purchased form Cell Signaling Technology. rabbit anti-ASC (Cat# YT0365) was purchased from ImmunoWay Biotechnology. Mouse anti-Phospho-α-synuclein (Cat# pSyn #64) and rabbit anti-IBA1 (Cat# 019-19741) were purchased from FUJIFILM Wako Pure Chemical Corporation. Mouse anti-α-synuclein (Cat# sc-12767) and mouse anti-GSDMD (Cat# sc-393581) was purchased from Santa Cruz Biotechnology. Rabbit anti-NF-κB p65 (Cat# ab16502), goat anti-IBA1 (Cat# ab5076) and rabbit anti-GSDMD (Cat# ab219800) were purchased from Abcam. Rabbit anti-IL18 (Cat# A1115) was purchased from ABclonal. Rabbit anti-tyrosine hydroxylase (Cat# AB152) was purchased from Merck Millipore.

Zheng R., Yan Y., Dai S., Ruan Y., Chen Y., Hu C., Lin Z., Xue N., Song Z., Liu Y., Zhang B, & Pu J. (2023). ASC specks exacerbate α‑synuclein pathology via amplifying NLRP3 inflammasome activities. Journal of Neuroinflammation, 20, 26.

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Immunofluorescence staining of NLRP3, SF3A2, NEDD4, and GSDMD

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For cytofluorescense staining, cells grown on glass coverslips were fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.1% Triton X-100 for 15 min. Cells on coverslips were incubated with normal goat serum for 2 h. After extensive washing in PBS, cells were incubated with primary mouse anti-NLRP3 (1:200, AdipoGen) and rabbit anti-SF3A2 (1:200, Abclonal), rabbit anti-NEDD4 (1:200, Proteintech), rabbit anti-GSDMD (1:200, Abcam) antibody overnight at 4 °C. After three washes with PBS, cells were incubated with secondary FITC-conjugated anti-ribbit IgG or FITC anti-rabbit IgG anti-body for an additional 2 h at room temperature. Stained cells were photographed under a fluorescence microscope (Olympus, Tokyo, Japan) with the B-2A (EX: 450–490, DM: 505, BA: 520) and G-2A (EX: 510–560, DM: 575, BA: 590) filters. The fluorescence of cells was quantified using ImageJ software (version 1.37; National Institutes of Health, Bethesda, MD, USA).

Sun W., Lu H., Cui S., Zhao S., Yu H., song H., Ruan Q., Zhang Y., Chu Y, & Dong S. (2023). NEDD4 ameliorates myocardial reperfusion injury by preventing macrophages pyroptosis. Cell Communication and Signaling : CCS, 21, 29.

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Immunoblotting of pro-IL-18 and GSDMD

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Processing of pro-IL-18 and GSDMD in cells was analyzed by immunoblotting using a rabbit anti-IL-18 antibody (MBL) or rabbit anti-GSDMD or rabbit anti-cleaved NT-GSDMD monoclonal antibodies (both from Abcam), respectively. THP1 macrophages were electroporated with LPS as described above. In order to capture proteins present in both the cell lysate and the cell culture supernatant, cells were resuspended in 1 ml of serum-free Opti-MEM instead of cRPMI. After incubation for 2 h, samples for immunoblotting were prepared by adding 250 μl of 5x SDS loading buffer directly to the well and heated to 65 °C for 10 min to fully denature proteins.

Devant P., Dong Y., Mintseris J., Ma W., Gygi S.P., Wu H, & Kagan J.C. (2023). Structural insights into cytokine cleavage by inflammatory caspase-4. Nature, 624(7991), 451-459.

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Molecular Mechanisms of Hippocampal Inflammation

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The hippocampus was weighed and homogenized in RIPA buffer containing protease inhibitors and phosphatase inhibitors. After the lysates were centrifuged at 12,000 g for 10 min at 4°C, supernatants were collected and protein concentrations were analyzed using bicinchoninic acid assay. Next, loading buffering 5X was added, and proteins were boiled for 5 min in a metal bath. Protein samples were run on SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, and then blocked with 3% BSA. The membranes were incubated with the following primary antibodies over night at 4°C: rabbit anti-SIRT3 (1:1000, CST), rabbit anti-p-NF-κB p65 (1:800, CST), rabbit anti-NF-κB p65 (1:1000, CST), rabbit anti-NLRP3 (1:800, Abcam), rabbit anti-ASC (1:1000, Abcam), rabbit anti-cleaved-Capspase-1 (1:1000, Abcam), rabbit anti-cleaved-IL-1β (1:1000, Abcam), rabbit anti-cleaved-IL-18 (1:1000, Abcam), rabbit anti-GSDMD-N (1:800, Abcam), rabbit anti-GSDMD (1:1000, Abcam), rabbit anti-PSD-95 (1:1000, CST), rabbit anti-Synapsin-1 (1:1000, CST), rabbit anti-GAPDH (1:1000, Abcam). HRP-conjugated Affinipure Goat Anti-Rabbit IgG (1:5000, proteintech) were incubated for 90 min at room temperature. The membranes were digitally scanned by an imaging system (Bio-Rad, USA) and then quantified using ImageJ software. The relative expression levels of all proteins were normalized to GAPDH. n=6 per group.

Xia B., Tong Y., Xia C., Chen C, & Shan X. (2020). α-Cyperone Confers Antidepressant-Like Effects in Mice via Neuroplasticity Enhancement by SIRT3/ROS Mediated NLRP3 Inflammasome Deactivation. Frontiers in Pharmacology, 11, 577062.

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