Total protein lysates from the kidney or podocytes were solubilized in SDS-PAGE sample buffer, separated on a 10%–12% SDS-polyacrylamide gel, and transferred electrophoretically onto polyvinylidenedifluoride (PVDF) membranes. Membranes were incubated for 1 h at 24 °C in Tris-buffer saline (TBS)-0.1% Tween-20 containing 5% BSA. Membranes were then incubated overnight at 4 °C with the primary antibodies to desmin (1:500) (Santa Cruz), synaptopodin (1:500) (Santa Cruz), NLRP3 (1:300) (Abcam), cleaved caspase-1 (1:500) (Cell signaling technology), cleaved gasdermin D (1:500) (Cell signaling technology), TXNIP (1:400) (Abcam), EGR1 (1:500) (Cell signaling technology), EZH2 (1:500) (Cell signaling technology), H3K27me3 (1:500) (Cell signaling technology), β-actin (1:2000) (Abcam), and histone H3 (1:200) (Cell signaling technology). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 24 °C, and developed using a chemiluminescent substrate (Pierce). Band intensities were quantified using Image J software.
Cleaved gasdermin d
Manufactured by Cell Signaling Technology
Sourced in United States
Cleaved gasdermin D is a laboratory product offered by Cell Signaling Technology. It functions as a molecular marker, specifically indicating the cleavage of the gasdermin D protein, which is a key component in the process of pyroptosis, a form of programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 1, by Cell Signaling Technology (3 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Desmin, by Santa Cruz Biotechnology (1 mentions)
Synaptopodin, by Santa Cruz Biotechnology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Nlrp3, by Abcam (1 mentions)
Txnip, by Abcam (1 mentions)
Hybond ecl nitrocellulose membrane, by GE Healthcare (1 mentions)
Ripa lysis buffer system, by Santa Cruz Biotechnology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Aggrecan, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lamin b, by Abcam (1 mentions)
Dulbecco s modified eagle s medium dmem ham s f12 medium, by Thermo Fisher Scientific (1 mentions)
Cox 2, by Cell Signaling Technology (1 mentions)
P iκbα, by Cell Signaling Technology (1 mentions)
Collagen 2, by Abcam (1 mentions)
4 protocols using cleaved gasdermin d
1
Protein Expression Analysis in Podocytes
2
Western Blot Analysis of Inflammation Markers
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Proteins from tissues and cells were purified with RIPA Lysis Buffer System (Santa Cruz, CA), and loaded onto 12% Mini-PROTEAN® TGX™ Precast Gel (Bio-rad, CA) for electrophoresis. The size-separated proteins will be then transferred to Hybond ECL Nitrocellulose Membranes (GE Healthcare, NJ). After blocking with 5% BSA buffer for 1 h, the membranes were incubated with primary antibody at 1:1000 dilution overnight at 4 °C. After washing with PBS containing 0.1% Tween-20, membranes were incubated with secondary antibody for 1 h and signals were detected with Pierce ECL Western Blotting Substrate (Thermo Scientific, IL). Intensity quantification of the bands were obtained with Image J software and normalized to β-actin.
Antibodies directed at PCSK9 (Cat. ab31762), NLRP3 (Cat. ab214185) and IL-1β (Cat. ab9722) were purchased from Abcam (San Francisco, CA). Antibody directed at p-JNK1 was purchased from Thermo Scientific (Cat. PA5-37698) and JNK1 antibody was purchased from Santa Cruz (Cat. sc-1648). Antibodies directed at p-ERK (Cat. 8544), ERK (Cat. 4695), p-P38 (Cat. 4511), P38 (Cat. 8690) and Cleaved Gasdermin D (Cat. 50928) were purchased from Cell signaling (Danvers, MA).
Antibodies directed at PCSK9 (Cat. ab31762), NLRP3 (Cat. ab214185) and IL-1β (Cat. ab9722) were purchased from Abcam (San Francisco, CA). Antibody directed at p-JNK1 was purchased from Thermo Scientific (Cat. PA5-37698) and JNK1 antibody was purchased from Santa Cruz (Cat. sc-1648). Antibodies directed at p-ERK (Cat. 8544), ERK (Cat. 4695), p-P38 (Cat. 4511), P38 (Cat. 8690) and Cleaved Gasdermin D (Cat. 50928) were purchased from Cell signaling (Danvers, MA).
3
Molecular Mechanisms of Inflammatory Osteoarthritis
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Chengdu Munster Co., Ltd. (Chengdu, China) supplied Sipeimine (purity ≥98 %). Dojindo (Kumamo, Japan) supplied the Cell-Counting Kit-8 (CCK-8) assays. Primary antibodies targeting proteins IκBα (#ab32518), MMP-13 (#ab39012), ASC (#ab309497), NLRP3 (#ab263899), caspase-1 (#ab138483), aggrecan (#ab216965), ADAMTS-5 (#ab41037), Lamin B (#ab16048), collagen-II (#ab34712) were meticulously procured from Abcam (Cambridge, MA, USA). Sigma Chemical Co. (St. Louis, MO, USA) supplied collagenase II. Additional primary antibodies p65 (#8242), p-p65 (#3033), Gasdermin D (#39754), cleaved-Gasdermin D(#10137), cleaved-caspase-1 (#89332), p-IκBα (#9246), PI3K (#4249), P-PI3K (#4228), P-AKT (#9271), AKT (#9272), COX-2 (#12282), iNOS (#13120), GAPDH (#2118) were contributed by Cell Signaling Technology (MA, USA). For immunofluorescence labeling, secondary antibodies (Alexa Spectrum®594-conjugated and Alexa Spectrum®488-tagged) were chosen from Jackson ImmunoResearch (West Grove, PA, USA). Essential enzyme-linked immunosorbent assay (ELISA) kits of IL-1β (#MLB00C), IL-18 (#DY7625-05), nitrite (#KGE001) and PGE2 (#KGE004B) were furnished by R&D Systems (Minneapolis, MN, USA), and Fetal Bovine Serum (FBS) along with Dulbecco's Modified Eagle's Medium (DMEM)/Ham's F12 medium were provided by Gibco (Grand Island, NY, USA).
4
Protein Expression and Quantification
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The proteins were isolated with RIPA lysis buffer (Beyotime), and the protein concentration was tested using the BCA protein kit (Beyotime). Following that, total protein (20 μg) was separated using 10% SDS‐PAGE and transferred to a Millipore PVDF membrane. The membranes were then blocked and incubated with the primary antibodies against KLF4 (abcam, Cambridge, UK, 1:1000, #ab129473), TRPV1 (abcam, 1:1000, #ab203103), NLRP3 (abcam, 1:1000, # ab263899), Cleaved caspase 1 (Cell Signalling Technology, MA, USA, 1:1000, #89332), Cleaved Gasdermin‐D (Cell Signalling Technology, 1:1000, #34667), AKT (1:500, # ab8805), p‐AKT (1:1000, # ab38449) and β‐actin (1:5000, # ab6276) overnight at 4°C. Subsequently, the membranes were incubated with the HRP‐conjugated secondary antibody for 1 h. The blots were visualized by the ECL kit (Beyotime) using the Gel imager (Biorad, CA, USA). The densitometry analysis was performed using Image J.
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