GSDMD Antibody - #AF4013

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Product: GSDMD Antibody
Catalog: AF4013
Description: Rabbit polyclonal antibody to GSDMD
Application: WB IF/ICC
Reactivity: Human, Mouse
Mol.Wt.: 50-53 kDa; 53kD(Calculated).
Uniprot: P57764
RRID: AB_2846780

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IF/ICC Protocol

Product Info

Source:
Rabbit
Application:
WB 1:500-1:2000, IF/ICC 1:100-1:500
*The optimal dilutions should be determined by the end user.
*Tips:

WB: For western blot detection of denatured protein samples. IHC: For immunohistochemical detection of paraffin sections (IHC-p) or frozen sections (IHC-f) of tissue samples. IF/ICC: For immunofluorescence detection of cell samples. ELISA(peptide): For ELISA detection of antigenic peptide.

Reactivity:
Human,Mouse
Clonality:
Polyclonal
Specificity:
GSDMD Antibody detects endogenous levels of total GSDMD.
RRID:
AB_2846780
Cite Format: Affinity Biosciences Cat# AF4013, RRID:AB_2846780.
Conjugate:
Unconjugated.
Purification:
The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).
Storage:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
Alias:

Fold/Unfold

1810036L03Rik; DF 5L; DF5L; DFNA 5L; DFNA5L; FKSG 10; FKSG10; FLJ12150; Gasdermin D; Gasdermin domain containing 1; Gasdermin domain containing protein 1; Gasdermin domain-containing protein 1; Gasdermin-D; GasderminD; GSDMD; GSDMD_HUMAN; GSDMDC 1; GSDMDC1;

Immunogens

Immunogen:

A synthesized peptide derived from human GSDMD.

Uniprot:

P57764(GSDMD_HUMAN) >>Visit HPA database.

Gene(ID):
GSDMD(79792)
Expression:
P57764 GSDMD_HUMAN:

Expressed in the suprabasal cells of esophagus, as well as in the isthmus/neck, pit, and gland of the stomach, suggesting preferential expression in differentiating cells.

Sequence:
MGSAFERVVRRVVQELDHGGEFIPVTSLQSSTGFQPYCLVVRKPSSSWFWKPRYKCVNLSIKDILEPDAAEPDVQRGRSFHFYDAMDGQIQGSVELAAPGQAKIAGGAAVSDSSSTSMNVYSLSVDPNTWQTLLHERHLRQPEHKVLQQLRSRGDNVYVVTEVLQTQKEVEVTRTHKREGSGRFSLPGATCLQGEGQGHLSQKKTVTIPSGSTLAFRVAQLVIDSDLDVLLFPDKKQRTFQPPATGHKRSTSEGAWPQLPSGLSMMRCLHNFLTDGVPAEGAFTEDFQGLRAEVETISKELELLDRELCQLLLEGLEGVLRDQLALRALEEALEQGQSLGPVEPLDGPAGAVLECLVLSSGMLVPELAIPVVYLLGALTMLSETQHKLLAEALESQTLLGPLELVGSLLEQSAPWQERSTMSLPPGLLGNSWGEGAPAWVLLDECGLELGEDTPHVCWEPQAQGRMCALYASLALLSGLSQEPH

PTMs - P57764 As Substrate

Site PTM Type Enzyme
Y37 Phosphorylation
K43 Ubiquitination
K51 Ubiquitination
K55 Ubiquitination
K62 Ubiquitination
S79 Phosphorylation
K145 Ubiquitination
S152 Phosphorylation
Y158 Phosphorylation
T161 Phosphorylation
K168 Ubiquitination
S181 Phosphorylation
S185 Phosphorylation
S201 Phosphorylation
K203 Ubiquitination
K204 Ubiquitination
K236 Ubiquitination
K248 Ubiquitination
S250 Phosphorylation
T251 Phosphorylation
S252 Phosphorylation
S261 Phosphorylation
K299 Ubiquitination

Research Backgrounds

Function:

Promotes pyroptosis in response to microbial infection and danger signals. Produced by the cleavage of gasdermin-D by inflammatory caspases CASP1 or CASP4 in response to canonical, as well as non-canonical (such as cytosolic LPS) inflammasome activators. After cleavage, moves to the plasma membrane where it strongly binds to inner leaflet lipids, including monophosphorylated phosphatidylinositols, such as phosphatidylinositol 4-phosphate, bisphosphorylated phosphatidylinositols, such as phosphatidylinositol (4,5)-bisphosphate, as well as phosphatidylinositol (3,4,5)-bisphosphate, and more weakly to phosphatidic acid and phosphatidylserine. Homooligomerizes within the membrane and forms pores of 10 - 15 nanometers (nm) of inner diameter, possibly allowing the release of mature IL1B and triggering pyroptosis. Exhibits bactericidal activity. Gasdermin-D, N-terminal released from pyroptotic cells into the extracellular milieu rapidly binds to and kills both Gram-negative and Gram-positive bacteria, without harming neighboring mammalian cells, as it does not disrupt the plasma membrane from the outside due to lipid-binding specificity. Under cell culture conditions, also active against intracellular bacteria, such as Listeria monocytogenes (By similarity). Strongly binds to bacterial and mitochondrial lipids, including cardiolipin. Does not bind to unphosphorylated phosphatidylinositol, phosphatidylethanolamine nor phosphatidylcholine.

PTMs:

Cleavage at Asp-275 by CASP1 (mature and uncleaved precursor forms) or CASP4 relieves autoinhibition and is sufficient to initiate pyroptosis. Cleavage at Asp-87 by CASP3.

Subcellular Location:

Cytoplasm>Cytosol. Inflammasome.
Note: In response to a canonical inflammasome stimulus, such as nigericin, recruited to NLRP3 inflammasone with similar kinetics to that of uncleaved CASP1 precursor.

Cell membrane. Secreted.
Note: Released in the extracellular milieu following pyroptosis.

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
Tissue Specificity:

Expressed in the suprabasal cells of esophagus, as well as in the isthmus/neck, pit, and gland of the stomach, suggesting preferential expression in differentiating cells.

Subunit Structure:

In response to a canonical inflammasome stimulus, such as nigericin, recruited to NLRP3 inflammasone with similar kinetics to that of uncleaved CASP1 precursor. Although this recruitment is also observed in the absence of PYCARD, it is more efficient in its presence (By similarity). Gasdermin-D, N-terminal forms disulfide-linked homooligomers (16-mers) in a Ca(+2)-independent manner. Oligomerization occurs in the presence of membranes; cytosolic Gasdermin-D, N-terminal remains monomeric.

Family&Domains:

Intramolecular interactions between N- and C-terminal domains may be important for autoinhibition in the absence of cleavage by inflammatory caspases CASP1 or CASP4. The intrinsic pyroptosis-inducing activity is carried by gasdermin-D, N-terminal, that is released upon cleavage by inflammatory caspases.

Belongs to the gasdermin family.

Research Fields

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

References

1). Cyclic helix B peptide alleviates proinflammatory cell death and improves functional recovery after traumatic spinal cord injury. Redox Biology, 2023 (PubMed: 37290302) [IF=11.4]
2). Blockade of mIL-6R alleviated lipopolysaccharide-induced systemic inflammatory response syndrome by suppressing NF-κB-mediated Ccl2 expression and inflammasome activation. MedComm, 2022 (PubMed: 35548710) [IF=9.9]

Application: WB    Species: Human    Sample: THP‐1 cells

FIGURE 5 The IL‐6R mAbs alleviated pyroptosis of THP‐1 by regulating NLRP3‐mediated inflammasome formation. (A) Immunoblotting and densitometry analysis of NLRP3, GSDMD, Caspase‐1 in the THP‐1 cells treated with the h‐mIL‐6R mAb (5 and 20 μg/ml) or Tocilizumab (5 μg/ml) for 30 min in advance, followed by treating with LPS (1 μg/ml) for 6 h. (B) ELISA and statistical analysis of IL‐1β levels in the supernatant of the THP‐1 cells with the indicated treatments identical to (A). (C,D) Flow cytometry analysis of cell pyroptosis with the indicated treatments in the THP‐1 cells. (E) Immunoblotting and densitometry analysis of NLRP3, GSDMD, Caspase‐1 in the THP‐1 cells treated with the h‐mIL‐6R mAb (10 μg/ml) or the NF‐κB inhibitor BAY 11–7082 (5 μM) for 30 min in advance, followed by treating with LPS (1 μg/ml) for 6 h. (F) ELISA and statistical analysis of IL‐1β levels in the supernatant of the THP‐1 cells with the indicated treatments identical to (E). (G,H) Flow cytometry analysis of cell pyroptosis with the indicated treatments in the THP‐1 cells. Cl‐Caspase‐1, cleaved Caspase‐1. The results were presented as mean ± SD through three independent experiments. The protein levels of N‐GSDMD and cleaved Caspase‐1 were normalized to the corresponding total proteins. The results were analyzed by one‐way ANOVA. *p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance
3). Betulinic Acid Inhibits ROS-Mediated Pyroptosis in Spinal Cord Injury by Augmenting Autophagy via the AMPK-mTOR-TFEB Signaling Pathway. International Journal of Biological Sciences, 2020 (PubMed: 33867836) [IF=9.2]

Application: IF/ICC    Species: Mice    Sample: spinal cords

Figure 2 BA attenuates pyroptosis after SCI. (A) Immunofluorescence staining for Caspase-1 and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (B) The quantitative mean optical density of the Caspase-1 in motor neurons of spinal cord lesion. (C) Immunofluorescence staining for GSDMD and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (D) The quantitative mean optical density of the GSDMD in motor neurons of spinal cord lesion. (E)Western blotting for ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels in the Sham, SCI, and BA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels were quantified and analyzed in each group. The values are expressed as the means ± SEM, n=5 per group. **p< 0.01, vs. Sham group. #p< 0.05 and ##p< 0.01, vs. SCI group.

Application: WB    Species: Mice    Sample: spinal cords

Figure 2 BA attenuates pyroptosis after SCI. (A) Immunofluorescence staining for Caspase-1 and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (B) The quantitative mean optical density of the Caspase-1 in motor neurons of spinal cord lesion. (C) Immunofluorescence staining for GSDMD and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (D) The quantitative mean optical density of the GSDMD in motor neurons of spinal cord lesion. (E)Western blotting for ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels in the Sham, SCI, and BA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels were quantified and analyzed in each group. The values are expressed as the means ± SEM, n=5 per group. **p< 0.01, vs. Sham group. #p< 0.05 and ##p< 0.01, vs. SCI group.
4). Cyclic helix B peptide promotes random‐pattern skin flap survival via TFE3‐mediated enhancement of autophagy and reduction of ROS levels. British Journal of Pharmacology, 2022 (PubMed: 34622942) [IF=7.3]

Application: IF/ICC    Species: Mouse    Sample: skin tissues

FIGURE 3 CHBP inhibits pyroptosis in random-pattern skin flaps. (a–d) Skin tissue sections were stained with anti-GSDMD-N (green) or anti-caspase 1 (CASP1; red) immunofluorescence antibodies (DAPI staining of the nuclei). Scale bar: 10 μm. The number of positive cells is shown on the right of representative images (n = 6 mice per group). (e,f) CHBP treatment reduced the expression of NLRP3, ASC, caspase 1 (CASP1), GSDMD-N, IL-1β and IL-18 in the context of ischaemia–reperfusion injury. Densitometric quantification is shown on the right (n = 6 mice per group). (g–j) CHBP treatment attenuated the activation of GSDMD, IL-1β, IL-18 and caspase 1 in the skin tissues detected by ELISA kits (n = 6 mice per group). Data shown are means ± SEM. *P 

Application: WB    Species: Mouse    Sample: skin tissues

FIGURE 3 CHBP inhibits pyroptosis in random-pattern skin flaps. (a–d) Skin tissue sections were stained with anti-GSDMD-N (green) or anti-caspase 1 (CASP1; red) immunofluorescence antibodies (DAPI staining of the nuclei). Scale bar: 10 μm. The number of positive cells is shown on the right of representative images (n = 6 mice per group). (e,f) CHBP treatment reduced the expression of NLRP3, ASC, caspase 1 (CASP1), GSDMD-N, IL-1β and IL-18 in the context of ischaemia–reperfusion injury. Densitometric quantification is shown on the right (n = 6 mice per group). (g–j) CHBP treatment attenuated the activation of GSDMD, IL-1β, IL-18 and caspase 1 in the skin tissues detected by ELISA kits (n = 6 mice per group). Data shown are means ± SEM. *P 
5). A snake cathelicidin enhances transcription factor EB-mediated autophagy and alleviates ROS-induced pyroptosis after ischaemia-reperfusion injury of island skin flaps. British journal of pharmacology, 2024 (PubMed: 37850255) [IF=7.3]
6). Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation. Neural regeneration research, 2024 (PubMed: 38227535) [IF=6.1]

Application: WB    Species: Rat    Sample:

Figure 6 BA alleviates pyroptosis and inflammation in the spinal cord tissue of SCI rats. On day 18 after SCI, Western blot assay was performed. (A–D) NLRP3 (A), ASC (B), GSDMD (C), and Caspase-1 (D) mRNA expression levels were measured by quantitative reverse transcription-polymerase chain reaction. (E) Western blot analysis of inflammation- and pyroptosis-associated protein expression levels. β-Actin was used as an internal reference for NLRP3, ASC, GADMD, IL-18, and IL-1β. GAPDH was used as an internal reference for TLR4, P65, p-P65, IκBα, and p-IκBα. (F–M) NLRP3 (F), ASC (G), GSDMD (H), IL-18 (I), IL-1β (J), TLR4 (K), p-P65/P65 (L), and p-IκBα/IκBα (M) protein expression levels were measured by Western blot assay. All western blot data were normalized to the sham group. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, vs. sham group; #P < 0.05, ##P < 0.01, vs. model group (one-way analysis of variance followed by Tukey's post hoc test). ASC: Apoptosis-associated speck-like protein containing CARD; BA: Biochanin A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; IL-18: interleukin-18; IL-1β: interleukin-1β; NLRP3: NOD-like receptor thermal protein domain associated protein 3; PC: positive control; p-P65: phospho-NF-κB P65; p-IκBα: phospho-NF-kappa-B inhibitor alpha; SCI: spinal cord injury; TLR4: Toll-like receptor 4.
7). Bexarotene improves motor function after spinal cord injury in mice. Neural Regeneration Research, 2023 (PubMed: 37449638) [IF=6.1]

Application: WB    Species: Mouse    Sample: spinal cord

Figure 3 Bexarotene attenuates neuronal pyroptosis in the spinal cord after SCI. (A) Immunofluorescence staining of Caspase 1 (pyroptosis-related marker, red) and NeuN (indicating neurons, green) (original magnification 30×) in spinal cord sections. The optical densities of Caspase 1 were markedly decreased in the SCI + Bex group and significantly increased in the SCI group. Scale bars: 25 μm. (B) Quantification of Caspase-1 in neurons of spinal cords in A. (C) Immunofluorescence staining of GSDMD-N (pyroptosis-related protein, green) and NeuN (indicating neurons, red) (original magnification 30×). Scale bars: 25 μm. (D) Quantification of GSDMD in neurons of spinal cords in C. (E–H) Evaluation of CASP1, GSDMD, IL-18, and IL-1β in the spinal cord by ELISA. (I) Western blot assay of GSDMD-N, NLRP3, Caspase-1, IL-1β, IL-18, and ASC. (J) Quantitative analyses of data from I; data were normalized to GAPDH. Data are expressed as the mean ± SEM (n = 6 mice per group). **P < 0.01, vs. sham group; ##P < 0.01, vs. SCI group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3- phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IL: interleukin; IOD: integrated optical density; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.
8). 贝沙罗汀改善脊髓损伤后运动功能的机制. 中国神经再生研究(英文版), 2023 (PubMed: 37449638) [IF=6.1]

Application: WB    Species: Mouse    Sample: spinal cord

Figure 3 Bexarotene attenuates neuronal pyroptosis in the spinal cord after SCI. (A) Immunofluorescence staining of Caspase 1 (pyroptosis-related marker, red) and NeuN (indicating neurons, green) (original magnification 30×) in spinal cord sections. The optical densities of Caspase 1 were markedly decreased in the SCI + Bex group and significantly increased in the SCI group. Scale bars: 25 μm. (B) Quantification of Caspase-1 in neurons of spinal cords in A. (C) Immunofluorescence staining of GSDMD-N (pyroptosis-related protein, green) and NeuN (indicating neurons, red) (original magnification 30×). Scale bars: 25 μm. (D) Quantification of GSDMD in neurons of spinal cords in C. (E–H) Evaluation of CASP1, GSDMD, IL-18, and IL-1β in the spinal cord by ELISA. (I) Western blot assay of GSDMD-N, NLRP3, Caspase-1, IL-1β, IL-18, and ASC. (J) Quantitative analyses of data from I; data were normalized to GAPDH. Data are expressed as the mean ± SEM (n = 6 mice per group). **P < 0.01, vs. sham group; ##P < 0.01, vs. SCI group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3- phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IL: interleukin; IOD: integrated optical density; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.

Application: IF/ICC    Species: Mouse    Sample: spinal cord

Figure 3 Bexarotene attenuates neuronal pyroptosis in the spinal cord after SCI. (A) Immunofluorescence staining of Caspase 1 (pyroptosis-related marker, red) and NeuN (indicating neurons, green) (original magnification 30×) in spinal cord sections. The optical densities of Caspase 1 were markedly decreased in the SCI + Bex group and significantly increased in the SCI group. Scale bars: 25 μm. (B) Quantification of Caspase-1 in neurons of spinal cords in A. (C) Immunofluorescence staining of GSDMD-N (pyroptosis-related protein, green) and NeuN (indicating neurons, red) (original magnification 30×). Scale bars: 25 μm. (D) Quantification of GSDMD in neurons of spinal cords in C. (E–H) Evaluation of CASP1, GSDMD, IL-18, and IL-1β in the spinal cord by ELISA. (I) Western blot assay of GSDMD-N, NLRP3, Caspase-1, IL-1β, IL-18, and ASC. (J) Quantitative analyses of data from I; data were normalized to GAPDH. Data are expressed as the mean ± SEM (n = 6 mice per group). **P < 0.01, vs. sham group; ##P < 0.01, vs. SCI group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3- phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IL: interleukin; IOD: integrated optical density; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.
9). Baicalein Attenuates Pyroptosis and Endoplasmic Reticulum Stress Following Spinal Cord Ischemia-Reperfusion Injury via Autophagy Enhancement. Frontiers in Pharmacology, 2020 (PubMed: 32903577) [IF=5.6]

Application: WB    Species: mouse    Sample: spinal cord

FIGURE 4 | Baicalein attenuates pyroptosis after spinal cord ischemia-reperfusion injury (SCIR).(C) Western blotting for NLRP3, GSDMD, and C-CASP1 expression levels in the Sham, SCIR+Vehicle, and SCIR+Baicalein groups. The gels were run under the same experimental conditions, and the cropped blots are shown here.
10). Rosuvastatin promotes survival of random skin flaps through AMPK-mTOR pathway-induced autophagy. International Immunopharmacology, 2023 (PubMed: 37001384) [IF=5.6]
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