Protein was extracted from the thoracic aorta of mice and HUVECs with RIPA lysis buffer containing protease inhibitor cocktail (Roche, Mannheim, Germany). Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with five percent skimmed milk at room temperature for 2–3 h and then incubated overnight at 4 °C with antibodies. The antibodies used were HDAC11 (1:250, Abcam, ab18973), GSDMD (1:1000, Cell Signaling Technology, #93709), GSDMD-N (1:1000, Cell Signaling Technology, #36425), GSDME (1:500, Proteintech, 13075-1-AP), GSDME-N (1:1000, Abcam, ab222407 and ab222408), TNFR1 (1:500, Wanleibio, Shenyang, China, WL01414), TNFR2 (1:500, Wanleibio, WL02956), pro-caspase-1 (1:1000, Wanleibio, WL02996), cleaved caspase-1 (1:1000, Wanleibio, WL02996a), NLRP3 (1:1000, Wanleibio, WL02635), ASC (1:1000, ImmunoWay Biotechnology Company, YT0365), pro-caspase-3 (1:500, Wanleibio, WL04004), cleaved caspase-3 (1:500, Wanleibio, WL01992), ERG (1:500, Proteintech, 14356-1-AP), and mouse anti-β-actin (1:1000, Cell Signaling Technology, #58169S). Secondary antibody and the enhanced chemiluminescent substrate were used for detection. Relative protein expression levels were semiquantitatively performed using Lane 1D software (Sage Creation Science Co, China).
Gsdmd n
Manufactured by Cell Signaling Technology
Sourced in United States, China
GSDMD-N is a recombinant protein that represents the N-terminal domain of the human Gasdermin D (GSDMD) protein. GSDMD is a key executor of pyroptosis, a form of inflammatory cell death. The N-terminal domain of GSDMD is responsible for the pore-forming activity that leads to cell lysis during pyroptosis.
Automatically generated - may contain errors
Lab products found in correlation
Il 18, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Nlrp3, by Abcam (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Mouse anti β actin, by Cell Signaling Technology (1 mentions)
Gsdmd, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Sirt1, by Abcam (1 mentions)
Caspase 1 p20, by Bioss Antibodies (1 mentions)
P ampk, by Abcam (1 mentions)
Il 1β, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Nlrp3, by Proteintech (1 mentions)
Bca kit, by Solarbio (1 mentions)
Cleaved caspase 1, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ecl kit, by Merck Group (1 mentions)
8 protocols using gsdmd n
1
Western Blot Analysis of Aorta and HUVEC Proteins
2
Myocardial Protein Extraction and Analysis
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The extraction of total myocardial tissue proteins was completed using RIPA lysis buffer (Solarbio, China), followed by the determination of protein concentration using a BCA kit (Solarbio, China). The proteins were denatured at high temperature, separated by SDS-PAGE electrophoresis, and then transferred to PVDF membranes (Millipore, USA). After completing the closure (60 min) at room temperature using non-fat milk, the proteins were incubated overnight at 4 ℃ with SIRT1 (dilution rate 1:1000, Abcam, USA), AMPK (dilution rate 1:1000, Abcam, USA), p-AMPK (dilution rate 1:1000, Abcam, USA), NLRP3 (dilution rate 1:1000, proteintech, China), GSDMD-N (dilution rate 1:1000, Cell Signaling Technology, USA), ASC (dilution rate 1:1000, Abcam, USA), IL-18 (dilution rate 1:1000, Abcam, USA), caspase-1 p20 (dilution rate 1:1000, Bioss, China), IL-1β (dilution rate 1:1000, Abcam, USA), and GAPDH (dilution rate 1:10000, Abcam, USA) primary antibodies. The proteins were incubated with secondary antibodies (dilution rate 1:10000, Santa Cruz Biotechnology, USA) at room temperature for 1 h the next day, and the protein bands were developed under the ultrasensitive automatic chemiluminescence imaging analysis system (FluorchemM, Protein Simple, USA). The detection of relative protein expression was completed using Image J (NIH, MD, USA) software.
3
Western Blot Analysis of Inflammasome Proteins
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Total protein was extracted from colonic tissue samples and THP-1 cells by homogenization in RIPA Lysis Buffer (cat. no. CW2333; CoWin Biosciences), as previously described (6 (link)). Protein concentration was determined using a bicinchoninic acid assay (Thermo Fisher Scientific, Inc.). Equal amounts of proteins (40 µg) were electrophoresed on 12% SDS-PAGE gels and subsequently transferred onto polyvinylidene difluoride membranes (Merck KGaA). Membranes were blocked with 5% BSA (Wuhan Goodbio Technology Co., Ltd.) at room temperature for 1 h, and then incubated with primary antibodies against: NLRP3 (cat. no. ab214185; Abcam), ASC (cat. no. 13833; Cell Signaling Technology, Inc.), cleaved caspase-1 (cat. no. 4199; Cell Signaling Technology, Inc.), GSDMD-N (cat. no. 93709; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 10494-1-AP; ProteinTech Group, Inc.) overnight at 4°C (all 1:1,000). Following the primary incubation, membranes were incubated with HRP-conjugated Affinipure Goat Anti-Rabbit/Mouse IgG secondary antibody for 1 h at room temperature (1:5,000; cat. nos. SA00001-2 and SA00001-2; ProteinTech Group, Inc.). Protein bands were visualized using the ECL kit (Merck KGaA). Densitometry data were analyzed using ImageJ (version 1.52V; National Institutes of Health).
4
Intestinal Protein Expression Analysis in CLP Mouse Model
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The mice were administered a strong anesthetic and slaughtered 16 h following the CLP procedure. The intestinal samples were swiftly collected and frozen at -80°C for future use. The samples were homogenized with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) at 12, 000 xg and 4°C for 20 min. To generate the standard curve, the supernatant was treated with a Bbicinchonininc acid (BCA) kit (Beyotime, Shanghai, China) and the absorbance was measured at 570 nm. The sample protein concentration was maintained at 10 g/l. Equal amounts of protein (20 mg) were separated by 10% SDS-PAGE and transferred to PVDF membranes (Invitrogen, USA) from several models. The membranes were blocked for 1 h at room temperature in blocking solution (5% BSA in TBST), then incubated overnight at 4°C with NLRP3 (1:1000, Abcam, USA), GSDMD (1:1000, Abcam, USA), GSDMD-N (1:1000, Cell Signaling, USA), IL-18 (1:1500, Abcam, USA), and beta-actin (1:5000, Cell Signaling, USA) antibodies. The sample was then treated with an IgG secondary antibody coupled with horseradish peroxidase for 1 h at room temperature. Using the ChemiDoc XRS technology, the protein bands were generated and photographed (Bio-Rad, USA). Using Image J, the relative density of the blots was measured.
5
Western Blot Analysis of NLRP3 Inflammasome Pathway
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For WB analysis, the mice were sacrificed and the peri-infarct tissues or the corresponding region of sham-operated mice were collected after 6 or 24 h of IPC and/or IPC + MCAO. Brain tissues were homogenized, and the total proteins were extracted. The cytosolic protein in samples was also extracted at 6 or 24 h after IPC and/or OGD. The protein concentration was determined with the Micro BCA Kit (Pierce Biotechnology, Rockford, IL, USA). The protein samples (50 μg) were subjected to 8–12% Tris-glycine SDS-PAGE and transferred to PVDF membranes with the electrophoretic transfer system. The membranes were then blocked with Tris-HCL buffer saline containing 10% skim milk and 0.1% Tween-20 (TBS-T) for 1 h at room temperature. Then, the membranes were incubated with primary antibody against NLRP3 (1:1000, #768319, Novus Biologicals, Centennial, CO, USA), ASC (1:1000, sc-514414, Santa Cruz, CA, USA), cleaved caspase-1 (1:1000, #AG-20B-0042-C, Adipogen, San Diego, CA, USA), and GSDMD-N (1:1000, #10137, Cell Signaling Technology, Danvers, MA, USA). Subsequently, the membranes were washed in TBS-T and incubated with secondary antibodies for 1 h at room temperature. Finally, immunoblots were scanned and analyzed via Image J program and normalized to a loading control β-actin (1:1000, #4970, Cell Signaling Technology, USA).
6
Western Blot Analysis of Protein Levels
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Western blotting assays were performed as previously described by us [27 (link), 28 (link)]. The following primary antibodies were obtained from Cell Signaling Technology (Danvers, MA): GSDMD-N (cat. #36425, dilution 1/2000), cleaved caspase-1 (cat. #4199, dilution 1/2000), NLRP3 (cat. #15101, dilution 1/1000), TOMM20 (cat. #42406, dilution 1/2000), COXIV (cat. #4850, dilution 1/2000), FOXO3a (cat. #2497, dilution 1/2000), and GAPDH (cat. #5174, dilution 1/3000). The Gel-Pro image program (Media Cybernetics, Las Vegas, USA) was used to measure the density of bands in a blinded manner. The protein level was expressed as a percentage of GAPDH to generate a relative protein level, and the relative protein level of the control group was expressed as a value of 1 (100%). Then, the protein levels of the other groups were normalized to this value. The obtained values were subjected to statistical analysis.
7
Western Blot Analysis of Inflammatory Proteins
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Total proteins were extracted from lung tissue homogenates using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The total protein concentrations were measured using an enhanced BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. Subsequently, the extracted protein samples were separated on 10–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to polyvinylidene fluoride (PVDF) membranes, and the PVDF membranes were blocked with 5% fat-free milk for 2 h. Then, the membranes were incubated with primary antibodies against NLRP3 (1:1,000, Abcam, Cambridge, United Kingdom), precursors of cysteinyl aspartate specific proteinase (procaspase)-1(1:500, Wanleibio, Shenyang, China) and cysteinyl aspartate specific proteinase (caspase-1) p20 (1:500, Wanleibio, Shenyang, China), gasdermin D (GSDMD), the N-terminal of GSDMD (GSDMD-N) (1:1,000, Cell Signaling Technology, United States), and β-actin (1:1,000, ZSGB-BIO, Beijing, China) at 4°C for 24 h. After three washes, the membranes were incubated with a peroxidase-conjugated secondary antibody (1:1,000, ZSGB-BIO, Beijing, China). Finally, the immune complexes were visualized with BeyoECL Plus (Beyotime Biotechnology, Shanghai, China), and the bands were quantified with ImageJ software.
8
Western Blot Analysis of Inflammasome Proteins
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Protein samples were loaded at 60–100 μg/well into a 10% SDS-PAGE gel for electrophoresis and transferred onto nitrocellulose filter membranes (PALL, New York, NY, USA), which were subsequently blocked in 5% non-fatted milk dissolved in PBS for 2 h. The blots were probed with specific primary antibodies against NLRP3 (1:1000, Cat. no. 15101; Cell Signaling Technology, Danvers, MA, USA), ASC (1:1000, Cat. no. 67824; Cell Signaling Technology, Danvers, MA, USA), GSDMD-N (1:500, Cat. no. 93709; Cell Signaling Technology, Danvers, MA, USA), and caspase-1 (1:500, Cat. no. 22915-1-AP; Proteintech, Chicago, IL, USA) and with GAPDH (1:1000, Cat. no. TA-08; ZSGB-BIO, Beijing, China) as an internal control. The membranes were then incubated on a shaker at 4 °C overnight with the primary antibody. The following day, membranes were incubated with horseradish-peroxidase-conjugated goat anti-mouse IgG or anti-rabbit IgG (1:5000; ZSGB-BIO, Beijing, China) for 1 h. Western blotting bands were quantified using the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) by measuring band intensity.
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