After adhesion, the total protein samples from the THP-1 cells were extracted with the RIPA lysis buffer (GenStar, China). BCA protein assay kit (GenStar) was adopted to measure the protein concentration. SDS-PAGE was performed with each lane containing 20 μg of protein samples. Separated proteins were transferred onto the PVDF membranes (Merck Millipore, Germany), followed by blocking in 5% non-fat milk powder and washing with TBST. The membranes were incubated overnight with diluted (1:1,000) primary antibodies, including anti-caspase 1 (#ab179515, Abcam, China), anti-cleaved-GSDMD (#36425, Cell Signaling Technology (CST), China), anti-NLRP3 (#15101, CST), and anti-β-actin (#C4, Diyibio, China), at 4°C. HRP-labeled anti-rabbit and anti-mouse IgG secondary antibodies (CST) were used for subsequent incubation of the membranes at room temperature for 1 h. The enhanced chemiluminescence (ECL) solution (Fdbio Science, China) was added to the membranes, followed by detection through a chemiluminescence imaging system (Baygene, China).
Anti cleaved gsdmd
Manufactured by Cell Signaling Technology
Sourced in China, United States
Anti-cleaved-GSDMD is a primary antibody product that detects the cleaved form of GSDMD (Gasdermin D), a protein involved in the process of pyroptosis, a type of programmed cell death. This antibody can be used in various applications to study the activation and regulation of the GSDMD protein.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by GenStar (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by GenStar (1 mentions)
Ab179515, by Abcam (1 mentions)
Ab71495, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti asc, by Santa Cruz Biotechnology (1 mentions)
Image lab 6, by Bio-Rad (1 mentions)
Anti nlrp3, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (1 mentions)
Anti il 1β, by Cell Signaling Technology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Bca assay, by Beyotime (1 mentions)
Chemidoctm xrs system, by Bio-Rad (1 mentions)
Electrophoretic transfer apparatus, by Bio-Rad (1 mentions)
Ab93876, by Abcam (1 mentions)
Image lab software system, by Bio-Rad (1 mentions)
Anti caspase 1 sc 56036, by Santa Cruz Biotechnology (1 mentions)
Anti caspase3 sc 56053, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh 6c5, by Merck Group (1 mentions)
4 protocols using anti cleaved gsdmd
1
Protein Expression Analysis of THP-1 Cells
2
Western Blot Analysis of Inflammatory Markers
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Tissues or cells were lysed in radioimmunoprecipitation assay buffer containing 1 mmol/L NaF, 1 mmol/L sodium orthovanadate, and 1 mmol/L phenylmethylsulfonyl fluoride. Protein concentrations were measured by BCA assay (Beyotime Biotechnology, China). Equal amounts of protein were separated by 4% to 20% GenScript’s SurePAGETM Bis‐Tris gels (GenScript Biotechnology, China) and transferred to polyvinylidene fluoride membranes (Millipore, USA). The following primary antibodies were used: anti RBP4 (Abcam, ab109193, UK), anti‐Caspase‐1 (Abcam, ab179515, UK), anti‐IL‐18 (Abcam, ab71495, UK), anti‐Tublin (Abcam, ab6046, UK), anti‐GSDMD (L60) (Cell Signaling, #93709, USA), anti‐NLRP3 (Cell Signaling, #15101, USA), anti‐IL‐1β (Cell Signaling, #12242, USA), anti‐β‐actin (Cell Signaling, #4970, USA), anti‐Cleaved GSDMD (Cell Signaling, #50928, USA), anti‐ASC (Santa Cruz, sc‐514414, USA). Chemiluminescence detection was performed after incubation with horseradish peroxidase‐conjugated secondary antibody (GE Healthcare, USA) using the ChemiDocTM XRS+ System (Bio‐Rad, USA). The intensity of immunoreactivity was assessed using Image Lab 6.0 software (Bio‐Rad, USA).
3
Western Blot Analysis of Protein Expression
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Western blot analysis of protein expression was performed as in our previously described protocol (36 (link)). Following quantification of protein lysates from BMMSCs, the samples (50 µg) were loaded on an SDS-PAGE gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes with use of an electrophoretic transfer apparatus (Bio-Rad Laboratories) overnight. PVDF membranes were blocked with 5% fat-free dry milk solution and incubated with primary antibodies (1:1,000 dilution, anti-FNDC5/Irisin antibody, no. ab131390; Abcam), anti-NLRP3 antibody (ab263899; Abcam); anti–caspase-1 (sc-56036; Santa Cruz Biotechnology); anti–cleaved GSDMD (50928; Cell Signaling Technology); anti–caspase-3 (sc-56053; Santa Cruz Biotechnology); anti-RUNX2 (sc-390351; Santa Cruz Biotechnology); anti-OCN (ab93876; Abcam), and anti-GAPDH 6C5 (CB1001; Merck Millipore) for 2 h at room temperature. The PVDF membranes were then incubated with secondary antibody conjugated with horseradish peroxidase (1:5,000) at room temperature for 1 h, followed by chemiluminescence detection (Clarity Max Western ECL Substrate; Bio-Rad Laboratories). The protein bands were quantitatively analyzed with an Image Lab Software system (Bio-Rad Laboratories).
4
Molecular Mechanisms of Pyroptosis Regulation
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The antibodies and reagents used in this study included anti-IL1R1 (Abclonal, A5727), anti-GAPDH (Proteintech, HRP-60004), anti-CASP4 (Affinity, AF5130), anti-cleaved CASP4 (Affinity, AF5373), anti-IGSF6 (Abclonal, A15128), anti-phospho-NF-κB pp65 (Servicebio, GB13025), anti-Bcl2 (Abclonal, A0208), anti-CASP3 (Abclonal, A2156), anti-IL1B (Abclonal, A16288), anti-NLRP3 (Affinity, DF7438), anti-ASC (Abcam, ab180799), anti-GSDMD (Cell signaling technology, 39754), anti-cleaved GSDMD (Cell signaling technology, 10137), anti-NF-κB p65 (Cell signaling technology, 3033), Goat anti-rabbit IgG-HRP (Proteintech, HRP60004), Goat anti-rabbit pp65 (Servicebio, GB21303), TUNEL (Servicebio, GB1501), DAPI (Servicebio, G1012), Anakinra (MCE, AMG-719), and Belnacasan (Selllock, also known VX-765, S2228).
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