Caspase 1

Manufactured by Cell Signaling Technology

Sourced in United States, China, United Kingdom

Caspase-1 is a protease enzyme that plays a key role in the inflammatory response and programmed cell death (apoptosis) processes. It is responsible for the proteolytic cleavage and activation of the proinflammatory cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18).

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Lab products found in correlation

126 protocols using caspase 1

Western Blot Analysis of Immune Proteins

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Cellular extracts were prepared using a lysis buffer (Intron, Daejeon, Korea). The cellular proteins were separated by gel electrophoresis and then transferred to nitrocellulose membranes. The membranes were incubated with primary antibody, and then sequentially incubated with peroxidase-conjugated secondary antibodies. The Western bands were obtained by enhanced chemiluminescence (Intron). The following primary antibodies were used: phospho-p65, phospho-IκBα, caspase-1 (Cell Signaling Technology, Beverly, MA, USA); IL-1β (Abcam, Cambridge, MA, USA); NLRP3 and ASC (AdipoGen, San Diego, CA, USA); actin (Sigma-Aldrich).

Zhang Q.L., Jiang R.H., Li X.M., Ko J.W., Kim C.D., Zhu M.J, & Lee J.H. (2019). Inhibition of Poly(I:C)-Induced Inflammation by Salvianolic Acid A in Skin Keratinocytes. Annals of Dermatology, 31(3), 279-285.

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Molecular Mechanisms of Anti-Inflammatory Effects

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Phorbol 12-myristate 13-acetate (PMA), lipopolysaccharide (LPS), monosodium urate (MSU), and colchicine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Neoastilbin (purity ≥ 98%) was purchased from Sichuan Victory Biological Technology Co., Ltd. (Chengdu, China). Fetal bovine serum (FBS) was purchased from Gibco (Scoresby, Australia). Penicillin–streptomycin solution was purchased from HyClone (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) was purchased from Biosharp Life Sciences (Beijing, China). Enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were purchased from MEIMIAN (Shanghai, China). NLRP3, ASC, Caspase-1, NF-κB p-p65, NF-κB p65, p-IKKα, IKKα, p-IκBα, IκBα, β-actin and Histone H3 antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Xu W., Li F., Zhang X., Wu C., Wang Y., Yao Y, & Xia D. (2022). The Protective Effects of Neoastilbin on Monosodium Urate Stimulated THP-1-Derived Macrophages and Gouty Arthritis in Mice through NF-κB and NLRP3 Inflammasome Pathways. Molecules, 27(11), 3477.

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Western Blot Analysis of NLRP3 Inflammasome

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Total proteins were extracted, and equal amounts of protein lysate extracts were separated by 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Nonspecific binding was blocked with 5% skim milk for 2 hrs at room temperature. Membranes were incubated overnight at 4°C with primary antibodies for NLRP3 (Cell Signaling Technology), Caspase-1 (Cell Signaling Technology), ASC, ICAM-1 and VCAM-1 (Santa Cruz Biotechnology), washed and then incubated for 2 hrs at room temperature with horseradish peroxide-conjugated anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology). Blots were visualized with enhanced chemiluminescent substrate (Pierce Biotechnology). Relative quantities of proteins were determined with a densitometer.

Wan Z., Fan Y., Liu X., Xue J., Han Z., Zhu C, & Wang X. (2019). NLRP3 inflammasome promotes diabetes-induced endothelial inflammation and atherosclerosis. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 12, 1931-1942.

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Western Blot Analysis of Inflammatory Markers

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Western Blot studies were carried out as described previously (45 (link)–47 (link)). Briefly, control and experimental cells were harvested, lysed in RIPA buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1mM EDTA, 1% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail (Calbiochem, Cocktail Set I), 1mM PMSF, and 0.2mM sodium orthovanadate. Protein concentration was determined using the Bio-Rad Protein Assay kit (PIERCE, Rockford, IL). Total protein lysed extracts (30 μg/lane) were loaded on a 10 % polyacrylamide (PAGE) premade gel (Bio-Rad, Hercules, CA) and after transferring onto PVDF membrane were processed for immunostaining with primary antibodies against APOL1 (anti-mouse, #66124-I-IG, Protein tech), NLRP3 (anti-mouse, #sc-66846; Santa Cruz); ASC (sc-22514-R; Santa Cruz Biotechnology), Caspase-1and Cleaved (C ) Caspase-1 (antirabbit #4199;1:700, Cell Signaling): followed by treatment with horseradish peroxidase-labeled appropriate secondary antibodies. The blots were developed using a chemiluminescence detection kit (PIERCE, Rockford, IL) and exposed to X-ray film (Eastman Kodak Co., Rochester, NY). Equal protein loading and the protein transfers were confirmed by immunoblotting for determination of GAPDH protein using a monoclonal GAPDH antibody (#SC-47724; 1:3000, Santa Cruz) performed on the same (stripped) western blots.

Jha A., Kumar V., Haque S., Ayasolla K., Saha S., Lan X., Malhotra A., Saleem M.A., Skorecki K, & Singhal P.C. (2019). Alterations in Plasma Membrane Ion Channel Structures Stimulate NLRP3 Inflammasomes Activation in APOL1 Risk Milieu. The FEBS journal, 287(10), 2000-2022.

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Western Blot Analysis of Inflammasome Activation

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PBMCs were lysed with 200 µL of radioimmunoprecipitation assay (RIPA) buffer (Thermo Scientific, USA) containing protease inhibitor (Roche, Germany). Then, 50 µg of total protein was loaded into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, and the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After sealing with 5% skimmed milk at 37 °C for 120 min, the PVDF membranes were incubated at 4 °C overnight with the primary antibodies against capase-1 p10 (Abcam, 1:1,000), IL-1β p17 (R&D Systems, Minneapolis, MN, USA; 1:1,000), NLRP3 (Cell Signaling, 1:1,000), caspase-1 (Cell Signaling, 1:1,000), ASC (Cell Signaling, 1:1,000), p-p65 (Cell Signaling, 1:1,000), and p-IκB α (Cell Signaling, 1:1,000) or Lamin-B (Cell Signaling, 1:1,000). Following that, the membranes were incubated with goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies at 37 °C for 60 min. An automatic digital gel image analysis system Bio-Rad CFX-96 (Bio-Rad, CA, USA) was employed to determine and analyze the band densities.

Tian Z.G., Yao M, & Chen J. (2020). Micheliolide alleviates ankylosing spondylitis (AS) by suppressing the activation of the NLRP3 inflammasome and maintaining the balance of Th1/Th2 via regulating the NF-κB signaling pathway. Annals of Translational Medicine, 8(16), 991.

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Quantitative Analysis of Skin Inflammasome

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Protein from human and rodent skin was extracted in RIPA buffer containing phosphatases and proteases inhibitor cocktails (Roche). Protein concentrations were determined by the BCA protein assay kit (Pierce, Mississauga, ON, Canada). Proteins were resolved by SDS-PAGE followed by western blotting using the following antibodies at 1:1000 concentration: Caspase-1 (Cell Signaling, MA), cleaved Caspase-1 (Cell Signaling, MA), IL1β (Cell Signaling, MA), cleaved IL1β (Cell Signaling, MA), and GAPDH (Cell Signaling, MA). Species appropriate secondary antibodies conjugated to horseradish peroxidase (BioRad, Mississauga, ON, Canada) were used and proteins visualized by enhanced chemiluminescence using the BioRad ChemiDoc MP Imaging System. Band intensities were detected, normalized and quantified with the ChemiDoc and Image Lab 5.0 software (BioRad Laboratories, Hercules, CA). Antibody concentrations are expressed relative to GAPDH.

Vinaik R., Abdullahi A., Barayan D, & Jeschke M.G. (2019). NLRP3 Inflammasome Activity is Required for Wound Healing After Burns. Translational research : the journal of laboratory and clinical medicine, 217, 47-60.

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Oxidative Stress Signaling Pathways

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Cell culture supplies including DMEM/F-12 media, RPMI 1640 media, penicillin/streptomycin, L-glutamine, fetal bovine serum (FBS), CM-H₂DCFDA dye and MitoSox® dye were purchased from Invitrogen. Rotenone, mouse anti-β actin antibody, mitoTEMPO, disuccinimidyl suberate (DSS), BSA lyophilized powder and acridine orange (AO) were purchased from Sigma-Aldrich. Lipopolysaccharide (E. Coli O111:B4) (LPS) and c-Abl antibody were purchased from EMD Millipore. Dasatinib (DAS), p-c-Abl (pY412), PKCδ, p-PKCδ (pY311), phospho-IκBα, iNOS, ASC, TOM20, lamin B, tubulin and p65 antibodies were purchased from Santa Cruz Biotechnology. Beclin1, NLRP3, caspase-1, LC3B, p-c-Abl (pY245), IL-1β and IL-18 antibodies were purchased from Cell Signaling Technology. TFEB antibody was Bethyl laboratories, Inc. Caspase 1 (p10) antibody was purchased from Adipogen. The mouse IL-1β and IL-18 ELISA kits from eBiosciences.

Lawana V., Singh N., Sarkar S., Charli A., Jin H., Anantharam V., Kanthasamy A.G, & Kanthasamy A. (2017). Involvement of c-Abl kinase in microglial activation of NLRP3 inflammasome and impairment in autolysosomal system. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, 12(4), 624-660.

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NLRP3 Inflammasome Activation Pathway

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Total protein from cell monolayers was harvested into M-PER lysis buffer (Thermo Fisher Scientific) and western blotting was performed to determine the expression of NLRP3 (Enzo life sciences), caspase-1, ASC, IL-1β (Cell Signaling Technologies) and IL-18 (MBL). Co-immunoprecipitation (Co-IP) was performed overnight at 4°C using NLRP3 antibody. Immunocomplexes were collected with Protein G Sepharose beads (GE Healthcare) for 1 h at 4°C prior to western blotting.

Ruiz P.A., Morón B., Becker H.M., Lang S., Atrott K., Spalinger M.R., Scharl M., Wojtal K.A., Fischbeck-Terhalle A., Frey-Wagner I., Hausmann M., Kraemer T, & Rogler G. (2016). Titanium dioxide nanoparticles exacerbate DSS-induced colitis: role of the NLRP3 inflammasome. Gut, 66(7), 1216-1224.

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Cellular Mechanisms of NLRP3 Inhibition

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The NLRP3 inhibitor MCC950, TNFα inhibitor Enbrel and aconitine were obtained from Shanghai Selleck Chemical Co., Ltd. (Shanghai, China) and Meilun Biotech. Co., Ltd. (Dalian, China). The primary antibodies against GAPDH, ULK1, TNFα, FADD, FasL, Cytochrome C, Caspase‐3 and ‐8, Bcl‐2, mTOR and LC3 were purchased from Proteintech Co., Ltd. (Wuhan, China). The primary antibodies against p62, p‐ULK1, IL‐1β, RIP1, RIP3, MLKL, Caspase‐1 and NLRP3 were purchased from Cell Signaling Tech. (Danvers, MA, USA). The foetal bovine serum and DMEM culture were obtained from GIBCO (NY, USA). DAPI and trypsin were purchased from Keygen Biotech (Nanjing, China). The H9c2 cell lines were transfected with siRNA by Lipofectamine 2000 (Invitrogen, USA) according to the manufacture's protocol. Scramble siRNA Ribobio Co., Ltd. (Guangzhou, China) and human BNIP3‐specific siRNA (Santa Cruz, Cat# sc‐37451) were transfected with a final concentration of 10 nmol/L to the transfection culture for 6 hours or cultured overnight. The other reagents were of analytical grade and used directly unless stated otherwise.

Peng F., Zhang N., Wang C., Wang X., Huang W., Peng C., He G, & Han B. (2019). Aconitine induces cardiomyocyte damage by mitigating BNIP3‐dependent mitophagy and the TNFα‐NLRP3 signalling axis. Cell Proliferation, 53(1), e12701.

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PKCδ-Targeting siRNA and Tanzisertib Protocol

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The PKCδ-specific siRNA and negative-control siRNA were designed and synthesized by GenePharma (Shanghai, China), and Lipofectamine^TM 3000 Reagent was purchased from Thermo Fisher. Tanzisertib (CC-930) was purchased from Sellectchem (Sellectchem, Houston, TX, USA) and dissolved in DMSO. Primary antibodies against c-Myc, CDK2, CDK6, Cyclin D2, P21^cip1, P27^kip1, PCNA, cytochrome C, PKCδ, PKCα, PKCη, PKCβ, PKCζ, IL-6, ERK1/2, p-ERK1/2, P38/MAPK, p-P38/MAPK and GSDME were obtained from Abcam (Cambridge, UK). Antibodies against Caspase-3, Caspase-8, cleaved Caspase-8, Caspase-9, cleaved Caspase-9, PARP, Bax, Bcl-2, Cyclin D1, Survivin, SAPK/JNK, p-SAPK/JNK, NF-κB/P65, p-NF-κB/P65, Caspase-1 and NLRP3, along with anti-mouse and anti-rabbit secondary antibodies, were purchased from Cell Signaling Technology (Beverly, MA, USA). FastStart Universal SYBR Green Master (Rox) was purchased from Sigma-Aldrich (Roche, Germany). Primers were prepared by Invitrogen (Shanghai, China). Other reagents were analytical-grade or guaranteed reagent commercial products, and were used without further purification, unless otherwise noted.

Zhang Y., Yang J., Wen Z., Chen X., Yu J., Yuan D., Xu B., Luo H, & Zhu J. (2020). A novel 3’,5’-diprenylated chalcone induces concurrent apoptosis and GSDME-dependent pyroptosis through activating PKCδ/JNK signal in prostate cancer. Aging (Albany NY), 12(10), 9103-9124.

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