To assess the protein composition and association of proteins in the inflammasome. RPMI cultures were lysed in 1 ml of Radio Immunoprecipitation Assay (RIPA) [1% NP‐40, 0.5% DOC (Deoxycholic acid; Sigma D6750; Sigma‐Aldrich), 0.1% SDS, 50 mM Tris.HCl, pH 8.0, 150 mM NaCl] with protease inhibitor mixture (Sigma‐Aldrich). About 2 mg of cell lysates were immunoprecipitated with 4 μg ASC‐antibody (rabbit monoclonal; Santa Cruz Biotechnology) and 50 μl protein G‐Agarose (p4691; Sigma‐Aldrich) in 4°C for 16 hrs with nutation, and then spin down the beads at 2.4 × g for 30 min. at 4°C. The pelleted beads were washed four times with PBS buffer (pH 7.3, 137 mM NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). After the last wash, spin down once more at 5000 r.p.m. for 5 min. at 4°C without adding any buffer followed by aspirating the residual buffer. Add 15–20 μl SDS loading buffer mix the buffer and bead with finger tickling, and then boil the mixture for 10 min. and spin down with 14,000 r.p.m. for 10–30 sec. before analysis by Western Blotting using antibodies against NALP3 (mouse monoclonal, 1:400 dilution; Adipogen), pro‐caspase‐1 (mouse monoclonal, 1:400 dilution; Santa Cruz Biotechnology), and ASC (rabbit monoclonal, 1:400 dilution; Santa Cruz Biotechnology).
Pro caspase 1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Pro-caspase-1 is a recombinant human pro-caspase-1 protein. Caspase-1 is a protease enzyme involved in the inflammatory response and programmed cell death. The pro-form of the enzyme requires activation to become catalytically active.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 1, by Santa Cruz Biotechnology (4 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Il 1β, by Santa Cruz Biotechnology (2 mentions)
Caspase 1, by Santa Cruz Biotechnology (2 mentions)
Caspase 1 p10, by Santa Cruz Biotechnology (2 mentions)
β actin, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Asc antibody, by Santa Cruz Biotechnology (1 mentions)
Phospho jak2, by Cell Signaling Technology (1 mentions)
Phospho akt, by Santa Cruz Biotechnology (1 mentions)
Pro il 1β, by Santa Cruz Biotechnology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Pierce enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
15 protocols using pro caspase 1
1
Inflammasome Protein Composition Analysis
2
NLRP3 Inflammasome Activation in Macrophages
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All culture materials were purchased from Gibco (Grand Island, NY). LY294002 (PI3K/Akt inhibitor) and rapamycin (p70S6K inhibitor) were purchased from Calbiochem (La Jolla, CA). Mouse monoclonal antibodies (mAB) against SREBP-1c, NLRP3, IL-1β, pro-IL-1β, caspase-1, pro-caspase-1, phospho-Akt, and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antibodies against phospho-p70S6K, p70S6K, phospho-JAK2, and JAK2 were purchased from Cell Signaling Technology (Beverly, MA). SREBP-1c, NLRP3, JAK2 siRNA and control siRNA were purchased from the National RNAi Core Facility in Academic Sinica, Taipei, Taiwan. The integrin β1-siRNA were purchased from Invitrogen (Carlsbad, CA). P. gingivalis lipopolysaccharide (Pg-LPS) was purchased from InvivoGen (San Diego, CA). Other chemicals of reagent grade were obtained from Sigma (St. Louis, MO).
3
Western Blot Analysis of Caspase-1
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Western blot analysis was performed as we described previously [40] (link). Briefly, total protein was extracted using lysis buffer with protein inhibitor after being washed in cold PBS. Protein concentrations were measured and resuspended to 2 μg/μl. Cell lysates were run on a SDS-PAGE gel at a voltage of 100 V for 2 h, transferred into polyvinylidene difluoride membrane at voltage of 100 V for 1 h, and blocked with 5% non-fat milk in TBST buffer for 30 min. Then, the membrane was incubated with primary antibodies against, pro-caspase-1, or cleaved caspase-1 (1:500 dilution, Santa Cruz) overnight at 4 °C, followed by incubation with a secondary antibody labeled with HRP for 1 h at room temperature. The membrane was developed with Kodak Omat film after being washed 3 times with TBST. β-actin (1:8000 dilution, Santa Cruz) was reported to serve as a loading control. The intensity of the bands was quantified using ImageJ 6.0 (NIH, Bethesda, MD, USA) [41] (link).
4
BMSC-Derived Extracellular Vesicle Profiling
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The lysates derived from BMSCs, BMSC-Exo, HS-BMSC-Exo, and the middle turns of the cochlear tissues were separated by 10% SDS-PAGE and transferred to PVDF membranes, which were further blocked with 5% non-fat milk and incubated with primary antibodies against HSP70, CD63, CD9, Alix, pro-caspase-1, cleaved caspase-1, NLRP3, ASC, and GAPDH (Santa Cruz). Peroxidase-conjugated secondary antibody (Sigma-Aldrich, 1:2000 dilution) was incubated for 2 h at room temperature, and a Pierce™ Enhanced Chemiluminescence (Thermo Scientific) was applied to obtain the chemiluminescence signal. GAPDH was utilized as an internal control to normalize the relative expression of interest genes with NIH-Image J1.51p 22.
5
Protocols for Mitochondrial Oxidative Stress
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Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), glutamine, penicillin-streptomycin, ATP, and Mito-TEMPO were bought from the Invitrogen/GIBCO BRL (Grand Island, NY, USA). LPS (from Escherichia coli 055: B5), Coenzyme Q0 (2,3-dimethoxy-5-methyl-1,4-benzoquinone), 2′,7′-dihydrofluorescein-diacetate (DCFH2-DA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetylcysteine (NAC), and cycloheximide were bought from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against NLRP3 were obtained from Biorbyt (Cambridge, UK). Antibodies against IL1β and Parkin were acquired from Abcam (Cambridge, UK). Antibodies against Nrf2, p70 S6 kinase, p-p70 S6 kinase, procaspase-1, GAPDH, and β-actin were procured from Santa Cruz (Heidelberg, Germany). Antibodies against p62, Beclin-1, Bax, mTOR, AKT, PI3K, p-mTOR, p-AKT, p-PI3K, and histone H3 were brought from Cell Signaling Technology Inc. (Danvers, MA). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Calbiochem (La Jolla, CA, USA). Antibodies against PINK1 were purchased from Genetic Technology Inc. (Miami, FL, USA). The rest of the chemicals were of the highest commercially available grade and supplied by either Sigma (St. Louis, MO, USA) or Merck (Darmstadt, Germany).
6
Western Blot Analysis of Inflammatory Markers
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Western blot analysis of cell and tissue lysates was performed as previously described [13 (link)]. Briefly, membranes were blocked with 5% nonfat dry milk in TBST for 1 h and incubated overnight at 4 °C with primary Abs against SOD2 (Cell Signaling, Danvers, MA), NLRP3, ASC, pro-caspase-1, cleaved caspase-1, ACTB and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membranes were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary Ab (Cell Signaling), and protein bands were detected by chemiluminescence using Immobilon Forte Western HRP substrate (Millipore WBLUF0500). Densitometry was performed using ImageJ software (NIH, Bethesda, MD). All experiments were performed in triplicate.
7
Kidney Biomarker Expression Profiling
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Kidney tissue lysates (40 μg) were run on 10% SDS-polyacrylamide gel electrophoresis, subsequently transferred to nitrocellulose membrane. Blots were blocked with 5% BSA and then exposed to the appropriate primary antibody in dilutions suggested from the commercial supplier. Primary antibody KIM-1, NGAL, TNF-α, IL-1β, IL-6, cryopyrin (NLRP3), ASC, pro-caspase-1, and β-actin were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Primary antibodies were probed with horseradish peroxidase conjugated goat anti-rabbit-IgG or anti-mouse-IgG. Visualization was performed by chemiluminescence (EzWestLumi plus, ATTO, NY, USA). Capturing image was achieved by gel image system (iBright FL100, Thermo Fisher Scientific, Waltham, MA, USA).
8
Antibody Detection in EMT Studies
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Antibodies against Zeb-1, horseradish peroxidase-conjugated donkey anti-rabbit and anti-mouse IgGs were purchased from Cell Signaling Technology (Beverly, MA). Antibodies recognizing E-cadherin and N-Cadherin were obtained from BD Bioscience (San Jose, CA). Antibodies against NLRP3 and ASC were purchased from Adipogen (Liestal, Switzerland). Antibodies targeting CD63, Rab5, vimentin, P2X7 and pro-Caspase-1 were supplied from Santa Cruz Biotech (Santa Cruz, Ca). Anti-snail antibody was purchased from Abcam (Cambridge, UK). Anti-actin antibody was obtained from Sigma (St. Louis, MO). Alexa Fluor 488 donkey anti-mouse IgG, Alexa Fluor 568 goat anti-rabbit IgG and Alexa Fluor 546 Phalloidin antibodies were purchased from Life Technologies (Gaithersburg, CA). sEV-depleted fetal bovine serum (FBS) and control FBS were purchased from Systembioscience (Mountain View, CA).
9
Immunoblotting for Caspase-1 and Cryopyrin
10
Western Blot Analysis of Protein Markers
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The tissue fragments were lysed in radioimmunoprecipitation assay buffer and then centrifuged for 5 min at 12,000 × g. The lysate was collected, and the protein concentration was measured using a test kit. Equal amounts of protein were denatured and separated on 10% SDS‒PAGE gels and then transferred to polyvinylidene difluoride membranes. When we performed WB, the blots were cut prior to hybridization with antibodies, and we developed strips of different proteins on the same PVDF film separately. The membranes were blocked in 5% nonfat milk for 2 h at room temperature and subsequently incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted the following proteins: NLRP3 (1:500), ɑ- tubulin (1:1000), GAPDH (1:3000) (Cell Signaling Technology, USA), ASC (1:200), procaspase-1, caspase-1 (1:500), p-PKC α (1:500) (Santa Cruz, USA), IL-1β, pro-IL-1β (1:1000), PKC α (1:1000), and occludin (1:2000) (Abcam, USA). Then, the membranes were incubated with secondary antibodies (1:5000) for 2 h at room temperature, and ECL Super Signal reagent (Millipore, USA) was used to detect protein bands. Image J software was used to measure the relative band density of different proteins on the scanned membrane [1 (link), 8 (link)].
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