Gasdermin d

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Gasdermin D is a laboratory product that functions as a pore-forming protein involved in cell death pathways. It plays a role in the regulation of inflammatory responses.

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Gasdermin D (GSDMD)

3 protocols using gasdermin d

Molecular Profiling of NLRP6 Pathway

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BMDMs or lungs were harvested at designated time points and homogenized in PBS containing 0.1% Triton X-100 (phosphatase and protease inhibitor cocktail added). After centrifugation the supernatants were used for immunoblotting. Total protein content in the supernatant was measured using a BCA protein assay kit (Thermofisher, NY) to ensure that equal amounts of proteins were loaded onto 10% SDS-PAGE gels. Proteins were transferred to polyvinylidene fluoride membrane according to the protocol provided by Bio-Rad. Appropriate primary antibodies against mouse NLRP6 (Sigma, MO), phospho-MLKL (Abcam, MA), RIP3, RIP1, P47^phox, P67^phox, gp91^phox, phospho-p38 MAPK, phospho-JNK, phospho-Stat3, caspase-8, GAPDH (Cell Signaling, MA), caspase-1 (Adipogen), and gasdermin-D (Santa Cruz, CA) were added to the membrane and incubated overnight at 4⁰ C. Appropriate secondary antibodies were used, and the films were developed using ECL plus western blot detection system (ThermoFisher, NY). IL-1β, TNF-α, IFN-γ, IL-1α, and IL-6 were measured in BALF supernatants by ELISA according to the manufacturer’s protocol (eBioscience, CA).

Ghimire L., Paudel S., Jin L., Baral P., Cai S, & Jeyaseelan S. (2018). NLRP6 negatively regulates pulmonary host defense in Gram-positive bacterial infection through modulating neutrophil recruitment and function. PLoS Pathogens, 14(9), e1007308.

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Comprehensive Protein Profiling in Cell Lysates

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Whole cell lysates (WCL) were prepared in lysis buffer containing 0.1% Triton, dithiothreitol, and protease inhibitor cocktail tablets (CPIC) (Roche Diagnostics, Indianapolis, IN) for 30min on ice and then spun at 16,000xg for 5min at 4°C. WCL samples were probed for caspase-3 (Santa Cruz Biotechnology, Dallas, TX), caspase-9 (Santa Cruz Biotechnology, Dallas, TX), caspase-8 (Santa Cruz Biotechnology, Dallas, TX), caspase-1 (Cell Signaling Technology, Danvers, MA), ICAM-1 (Santa Cruz Biotechnology, Dallas, TX), and VCAM-1 (Santa Cruz Biotechnology, Dallas, TX). Nuclear (Nuc) and cytoplasmic (Cyto) protein fractions were prepared by using the CelLytic NuCLEAR extraction kit (Millipore Sigma, Merck KGaM, Darmstadt, Germany) and probed for cyclinB1 (Santa Cruz Biotechnology, Dallas, TX), γH2AX (Cell Signaling Technology, Danvers, MA), and NF-κB-p65 (Abcam, Cambridge, UK). Protein extraction from serum-free supernatant media using an adopted methanol-mediated extraction protocol (Shi et al. 2012 (link)) was used to probe in conjunction with WCL or Nuc/Cyto fractions for pro-inflammatory molecules Gasdermin D (Santa Cruz Biotechnology, Dallas, TX), HGMB-1 (Cell Signaling Technology, Danvers, MA), IL-1α (Santa Cruz Biotechnology, Dallas, TX), IFNβ (Santa Cruz Biotechnology, Dallas, TX), and IL-1β (Cell Signaling Technology, Danvers, MA).

Fountain M.D., McLellan L.A., Smith N.L., Loughery B.F., Rakowski J.T., Tse H.Y, & Hillman G.G. (2019). Isoflavone-Mediated Radioprotection Involves Regulation of Early Endothelial Cell Death and Inflammatory Signaling in Radiation-Induced Lung Injury.. International journal of radiation biology, 96(2), 245-256.

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Immunofluorescence Analysis of Neuroinflammatory Markers

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Anesthetized mice were successively perfused intracardially with 0.9% sodium chloride and 4% paraformaldehyde (PFA). Mouse brains were soaked in 4% PFA for 12 h and then dehydrated by sucrose solutions with an ascending concentration gradient of 10, 20, and 30% at 4 °C. After fixation in optimal cutting temperature compound (Sakura Finetek, USA), the brains were sliced into 15-μm sections. Brain sections and neuron coverslips were fixed with 4% PFA for 20 min and then permeabilized with blocking buffer comprising 5% goat serum, 1% bovine serum albumin (BSA) and 0.3% Triton X-100 at room temperature for 1 h. The samples were incubated overnight with primary antibodies against LDLR (1:200, Santa Cruz Biotechnology, USA), NeuN (1:500, Abcam, UK), GFAP (1:500, Abcam, UK), Iba-1 (1:100, Abcam, UK), NLRP3 (1:200, Abcam, UK), ASC (1:200, Santa Cruz Biotechnology, USA), Caspase-1 (1:200, Santa Cruz Biotechnology, USA), and Gasdermin D (1:200, Santa Cruz Biotechnology, USA) at 4 °C, followed by incubation with appropriate Alexa Fluor-488/594-conjugated secondary antibodies (Jackson ImmunoResearch) and DAPI. Immunofluorescence images were captured with a microscope (Olympus MX51, Japan). The positive signals were analyzed using ImageJ software.

Sun R., Peng M., Xu P., Huang F., Xie Y., Li J., Hong Y., Guo H., Liu Q, & Zhu W. (2020). Low-density lipoprotein receptor (LDLR) regulates NLRP3-mediated neuronal pyroptosis following cerebral ischemia/reperfusion injury. Journal of Neuroinflammation, 17, 330.

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