Cells were lysed 4 h after incubation with serum-derived EVs from control and TBI patients with a lysis buffer containing protease and phosphatase inhibitor cocktail (Sigma, St. Louis, MO, USA) and resolved in 4–20% Tris-TGX Criterion gels (Bio-Rad, Hercules, CA, USA). Protein detection was performed using antibodies to caspase-1 (Novus, Centennial, CO, USA), ASC (Santa Cruz, Santa Cruz, CA, USA), AIM2 (Santa Cruz, Santa Cruz, CA, USA), GSDMD (Novus, Centennial, CO, USA), and HMGB1 (Millipore, Burlington, MA, USA). Chemiluminescent quantification was performed using Bio-Rad Image Lab (Bio-Rad, Hercules, CA, USA) and all data were normalized to β-actin (Sigma, St. Louis, MO, USA).
Caspase 1
Manufactured by Novus Biologicals
Sourced in United States
Caspase-1 is a cysteine protease enzyme that plays a crucial role in the inflammatory response and programmed cell death. It is involved in the activation of pro-inflammatory cytokines and the initiation of the apoptotic pathway.
Automatically generated - may contain errors
Lab products found in correlation
Il 1β, by Cell Signaling Technology (10 mentions)
β actin, by Merck Group (7 mentions)
Nlrp3, by Novus Biologicals (6 mentions)
Chemidoc touch imaging system, by Bio-Rad (5 mentions)
Caspase 11, by Novus Biologicals (5 mentions)
Un scan it gel 6, by Silk Scientific (4 mentions)
Criterion tgx stain free precasted gels, by Bio-Rad (4 mentions)
Nlrp3, by Cell Signaling Technology (4 mentions)
Nlrp1, by Novus Biologicals (4 mentions)
Il 18, by Abcam (3 mentions)
Caspase 8, by Novus Biologicals (3 mentions)
Gsdmd, by Novus Biologicals (2 mentions)
Lumiglo, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Gapdh, by Proteintech (2 mentions)
Gapdh, by Merck Group (2 mentions)
Pyrin, by Santa Cruz Biotechnology (2 mentions)
Caspase 3, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
28 protocols using caspase 1
1
Traumatic Brain Injury EV Proteome Analysis
2
Inflammasome Protein Detection Protocol
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Protein levels of inflammasome signaling proteins were determined in the cytosolic and mitochondrial fractions as described in [7 (link)]. Briefly, protein lysates were resolved in 10–20% Criterion TGX Stain-Free precasted gels (Bio-Rad), using antibodies (1:1000 dilution) to NLRC4 (Novus Biologicals, cat# NBP2–41124), caspase-1 (Novus Biologicals, cat# NB100–56565), caspase-11 (Novus Biologicals, cat# MAB8648), ASC (Santa Cruz, cat# sc-271054), IL-1β (Cell Signaling, cat# 12242S), IL-18 (Abcam, cat# ab71495), HSP60 (Cell Signaling, cat#12165) and β-actin (Sigma Aldric, cat# A5441). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Corporation). Chemilluminescence substrate (LumiGlo, Cell Signaling) was imaged with the ChemiDoc Touch Imaging System (BioRad).
3
Western Blot Analysis of NLRP3, Caspase-1, and Cleaved Caspase-1 in Macrophages and Lung Tissue
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Proteins were extracted from RAW264.7 macrophages and fresh lung tissue using a Total Protein Extraction Kit (Nanjing KeyGenBiotech, Co, Ltd, China). Protein concentrations were determined by BCA Assay Kit (Nanjing KeyGenBiotech, Co, Ltd, China). After separating the proteins electrophoretically on a with 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the total protein (30 μg/lane) was transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore Co, Ltd, Germany). The PVDF membranes were blocked with Tris‐buffered saline Tween containing 5% skimmed milk and incubated with rabbit monoclonal glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) (Jetway Biotech Co, Ltd, Guangzhou, China), NLRP3 (Cell Signaling Technology), caspase‐1 (Novus Biologicals, Centennial, CO), and cleaved caspase‐1 (Novus Biologicals) antibodies overnight at 4°C, and then incubated with a horseradish peroxidase‐labeled antirabbit secondary antibody (Jetway Biotech Co, Ltd, Guangzhou, China) for 1 hour. The blots were developed using an electrochemiluminescence color kit (Asbio Technology, Inc, Guangzhou, China), and densitometry was performed using Image J software. The relative protein expression levels of NLRP3, caspase‐1, and cleaved caspase‐1 were evaluated in triplicate using GAPDH as the internal standard.
4
Quantifying Cytokine Secretion and cAMP in Microglial Cells
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Cell-secreted cytokines were measured from the cell culture supernatant using commercially available ELISA kits for mouse TNF-α, IL-1β/IL-1F2 (R&D Systems, Minneapolis, MN, USA), IL-18 (MBL, Naka-Ku, Nagoya Aichi, Japan), and Caspase-1 (Novus Biologicals) according to the manufacturer’s protocols. BV2 cells were cultured in the presence of Aβ (2 µM), TDCA (400 ng/ml), or KH7 (4 μM) for 24 h. Cells were harvested and lysed with 0.1 M HCl to measure intracellular cAMP using a cAMP assay kit (Abcam) according to the manufacturer’s protocol.
5
Nicotine Exposure Alters Hippocampal and Cortical Proteins
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Rats exposed to nicotine (16–21 days) or saline were anesthetized using 5% isoflurane, decapitated, and the hippocampal and cortical tissues were collected, flash frozen, and stored at −80 °C. At the time of immunoblotting, hippocampal and cortical tissues were homogenized, protein content was analyzed, and proteins were separated by 12% SDS-PAGE as described [47 (link)]. Proteins were transferred to Immobilon-P (Millipore, Burlington, MA, USA) membrane and incubated with primary antibodies against rabbit polyclonal anti-ER-β (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), IL-1β (1:1000; Cell Signaling, Danvers, MA, USA), ASC (1:1000; Santa Cruz Biotechnology), Caspase-1 (1:1000; Novus Biologicals, Littleton, CO, USA), and β-Actin (1:5000; Sigma, St. Louis, MO, USA). All data were normalized to β-Actin (monoclonal; 1:1000; Sigma). Immunoblot images were digitized and subjected to densitometric analysis [45 (link)].
6
Inflammasome Protein Detection in CSF
7
Western Blot Analysis of Inflammatory Mediators
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Cells were lysed in ice-cold PBS containing a cocktail of protease and phosphatase inhibitors to isolate proteins. Protein concentration was determined according to the Bradford method [59 (link)] using γ-globulin as a standard.
Aliquots of 15 μg of protein were separated on 10% or 15% acrylamide gel by sodium-dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated overnight at 4 °C with specific primary antibodies against mPGES-1, IL-18 (Abcam®, Cambridge, UK), COX-2, iNOS, pJNK, JNK, P38, pP38, pERK1/2, ERK ½, Nrf-2, pSTAT-3, pJAK2, NLRP3, ASC, H3K18ac, H3K27me3, H3K9me3, H3 (Cell Signaling®, Danvers, MA, USA), HO-1 (Henzo®, Madrid, Spain), caspase-1 and caspase-11 (Novus Biologicals®, Littleton, CO, USA). Nitrocellulose membranes were incubated for 2 h in a blocking solution with antirabbit horseradish peroxidase-labeled (HRP) secondary antibody (Cell Signaling®, Danvers, MA, USA) or antimouse HRP secondary antibody (Dako®, Atlanta, GA, USA). β-Actin primary antibody (Abcam®, Cambridge, UK) was employed to demonstrate equal loading. The immunosignals were captured using Amersham Imager 600 from GE Healthcare® (Buckinghamshire, UK) and analyzed and quantified by Image Processing and Analysis in Java (ImageJ®, Softonic) and expressed comparing with the DMSO-LPS treated cells.
Aliquots of 15 μg of protein were separated on 10% or 15% acrylamide gel by sodium-dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated overnight at 4 °C with specific primary antibodies against mPGES-1, IL-18 (Abcam®, Cambridge, UK), COX-2, iNOS, pJNK, JNK, P38, pP38, pERK1/2, ERK ½, Nrf-2, pSTAT-3, pJAK2, NLRP3, ASC, H3K18ac, H3K27me3, H3K9me3, H3 (Cell Signaling®, Danvers, MA, USA), HO-1 (Henzo®, Madrid, Spain), caspase-1 and caspase-11 (Novus Biologicals®, Littleton, CO, USA). Nitrocellulose membranes were incubated for 2 h in a blocking solution with antirabbit horseradish peroxidase-labeled (HRP) secondary antibody (Cell Signaling®, Danvers, MA, USA) or antimouse HRP secondary antibody (Dako®, Atlanta, GA, USA). β-Actin primary antibody (Abcam®, Cambridge, UK) was employed to demonstrate equal loading. The immunosignals were captured using Amersham Imager 600 from GE Healthcare® (Buckinghamshire, UK) and analyzed and quantified by Image Processing and Analysis in Java (ImageJ®, Softonic) and expressed comparing with the DMSO-LPS treated cells.
8
Penumbra Protein Profiling for NLRP Inflammasome
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Protein samples were harvested from penumbra. Tissues were ground separately in RIPA buffer comprising protease and phosphatase inhibitors (cocktails and PMSF from Aspen) for 30 min at 4°C. A BCA kit (Aspen) was used to detect the total protein concentration of each sample. Proteins were processed by SDS-PAGE (10–12.5%) and electro-blotted onto a PVDF membrane. And the membrane was then incubated in blocking buffer (5% skim milk) for 1 h at room temperature and incubated with primary antibodies including GSDMD (Abclonal), NLRP1, NLRP3, Caspase-1 (Novus), IL-1β,IL-18 (R&D), total p-65 (Proteintech),phosphorylated p-65 (Abclonal), GAPDH (Proteintech) overnight at 4°C. After washing three times, the membrane was incubated in secondary antibody for 1 h at 24°C. The proteins were scanned with a Bio-Rad system. ImageJ software was used to quantify protein levels which were normalized to GAPDH (n = 6/group).
9
Inflammasome Protein Expression Analysis
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Analyses of inflammasome protein expression were measured by immunoblot analysis as described in Raval et al. (2019) (link). Briefly, cortical lysates were resolved in 4–20% Criterion TGX Stain-Free precasted gels (Bio-Rad, Hercules, CA, United States), using antibodies (1:1,000 dilution) to NLRP1 (Novus Biologicals, Littleton, CO, United States), caspase-1 (Novus Biologicals, Littleton, CO, United States), ASC (Santa Cruz, Dallas, TX, United States), IL-1β (Cell Signaling Technology, Danvers, MA, United States), caspase-8 (Novus Biologicals, Littleton, CO, United States), caspase-11 (Novus Biologicals, Littleton, CO, United States), and β-actin (Sigma-Aldrich, St. Louis, MO, United States). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Inc., Orem, UT, United States) and membranes were imaged using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, United States) following chemiluminescence.
10
Quantifying Inflammasome Activation in Spinal Cord
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