Cell lysates were extracted from infected macrophages in radioimmunoprecipitation assay (RIPA) buffer with complete protease inhibitor, phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail (all from Sigma). Cell culture supernatants were precipitated by methanol and chloroform to obtain protein samples as previously described [24 (link)]. Briefly, the supernatants were mixed with an equal volume of cold methanol and 1/4 volume of cold chloroform and then centrifuged at 13,000× g for 10 min at 4 °C. The upper phase was discarded, 1 ml of methanol was added, and the mixture was then centrifuged for 10 min at 13,000× g. The protein pellets were dried at room temperature and dissolved in 1% SDS buffer. The protein samples were separated and analyzed by SDS-PAGE and immunoblot. Membranes were blocked in 5% skim milk, incubated overnight at 4 °C with primary antibodies and visualized with secondary HRP-conjugated antibodies (Proteintech, Wuhan, China). The primary antibodies used were anti-mouse IL-1β (AF-401, R&D, Minneapolis, USA), anti-mouse caspase-1 (p20) (AG-20B-0042, Adipogen, Liestal, Switzerland), anti-NLRP3 (AG-20B-0014, Adipogen, Liestal, Switzerland) and anti-mouse β-actin (60008–1, Proteintech, Wuhan, China).
Ag 20b 0042
Manufactured by Adipogen
Sourced in United States, Switzerland
The AG-20B-0042 is a piece of lab equipment designed for general laboratory use. It is a compact and versatile device that can be utilized in a variety of research and testing applications. The core function of this product is to provide a controlled environment for various experiments and sample processing tasks.
Automatically generated - may contain errors
Lab products found in correlation
Ag 20b 0014, by Adipogen (18 mentions)
Sc 22514 r, by Santa Cruz Biotechnology (10 mentions)
Nigericin, by Merck Group (10 mentions)
Anti mouse caspase 1, by Adipogen (9 mentions)
Adenosine triphosphate (atp), by Merck Group (9 mentions)
Anti asc, by Santa Cruz Biotechnology (8 mentions)
Mouse anti il 1β, by Cell Signaling Technology (7 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Mcc950, by Targetmol (7 mentions)
Anti nlrp3, by Cell Signaling Technology (7 mentions)
Anti gapdh, by Proteintech (7 mentions)
Ab209845, by Abcam (5 mentions)
Pam3csk4, by InvivoGen (5 mentions)
Lps escherichia coli 055 b5, by Merck Group (5 mentions)
Epimedin a, by Targetmol (5 mentions)
Epimedin a1, by Targetmol (5 mentions)
Epimedin b, by Targetmol (5 mentions)
Epimedin c, by Targetmol (5 mentions)
Icariin, by Targetmol (5 mentions)
Anhydroicaritin, by Targetmol (5 mentions)
57 protocols using ag 20b 0042
1
Protein Extraction and Analysis from Infected Macrophages
2
Protein Extraction and Immunoblotting Protocol
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Cells were lysed in RIPA lysis buffer (20 mM Tris-HCl, pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, 2 mM ethylenediaminetetraacetic acid [EDTA], 200 nM aprotinin, 20 μM leupeptin, 50 mM phenanthroline, and 280 mM benzamidine-HCl) and supernatant fractions were collected. Protein concentrations were measured with bicinchoninic acid (Pierce, Rockford, IL, USA), and equal amounts of proteins (50 μg/lane) were separated by 13% sodium dodecyl-sulfate polyacrylamide gel electrophoresis and transferred to the nitrocellulose membranes (GE Healthcare Life Science, Pittsburgh, PA, USA) incubated with a specific antibody. The anti-caspase-1 antibody (3019-100; BioVision, Milpitas, CA, USA, and AG-20B-0042; AdipoGen, San Diego, CA, USA) and anti-GSDMD antibody (ab209845; Abcam PLC) were used as primary antibodies. These antibodies were used at a dilution of 1:1,000. The anti−ACTB antibody (A5441) was acquired from Sigma−Aldrich (Merck KGaA) and used at a dilution of 1:5000. Secondary antibodies (anti−rabbit IgG [1:2000 diluted; sc−2004] and anti−mouse IgG [1:2000 diluted; sc−2005]) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Bands were detected using the Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Darmstadt, Germany).
3
Western Blot Analysis of Inflammasome Markers
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Purified DCs (1×106) were lysed with 100-μl protein extraction reagent (89900, Thermo Scientific) containing protease inhibitor (5872S, Cell Signaling). Equal amounts of protein (lysate) or supernatants were run on an SDS-PAGE and transferred onto nitrocellulose membranes following electro blotting. After blocking with 5% fat-free milk, the membranes were incubated at 4°C overnight with the following primary antibodies: anti mouse pro & cleaved IL-1β (Cell Signaling, 12507), pro-caspase-1 (Abcam, ab108362), Caspase-p20 & 10 (Adipogen-AG-20B-0042) as per manufacturer's instructions. The membranes were then washed was incubated with Horseradish peroxidase–conjugated secondary antibody (Cell Signaling). Proteins were visualized with SuperSignal West Pico chemiluminescent substrate (34078, Thermo Scientific).
4
Immunoblotting Antibody Protocol
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Antibodies for immunoblotting include: rabbit anti-mouse ASC (sc-22514-R; Santa Cruz, CA, USA), mouse anti-mouse caspase-1 (AG-20B-0042; Adipogen, San Diego, CA, USA), rabbit anti-mouse mature and pro–IL-1β (sc-7884; Santa-Cruz, CA, USA) and mouse anti-mouse β-actin (sc-47778; Santa-Cruz, CA, USA).
5
Western Blot Analysis of Caspase-1 and IL-1β
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Culture samples were denatured in loading buffer containing SDS and 100
mM DTT, and boiled for 5 min. SDS-PAGE–separated proteins were
transferred to polyvinylidene difluoride membranes and immunoblotted with
primary Abs against caspase-1 (Adipogen; AG-20B-0042 or kind gift of Dr Peter
Vandenabeele, Ghent University), IL-1β (R&D Systems), and GAPDH
(Cell Signaling Technology; D16H11), followed by secondary anti-rabbit,
anti-rat, anti-mouse, or anti-goat HRP Abs (Jackson ImmunoResearch
Laboratories)54 (link).
Images have been cropped for presentation. Full size images are presented in
Supplementary Fig.
7 .
mM DTT, and boiled for 5 min. SDS-PAGE–separated proteins were
transferred to polyvinylidene difluoride membranes and immunoblotted with
primary Abs against caspase-1 (Adipogen; AG-20B-0042 or kind gift of Dr Peter
Vandenabeele, Ghent University), IL-1β (R&D Systems), and GAPDH
(Cell Signaling Technology; D16H11), followed by secondary anti-rabbit,
anti-rat, anti-mouse, or anti-goat HRP Abs (Jackson ImmunoResearch
Laboratories)54 (link).
Images have been cropped for presentation. Full size images are presented in
7
6
Immunohistochemical Analysis of GFAP and Casp-1
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Brain slices were treated in 0.03% H2O2 in 0.01M PBS for 15 minutes. After washing, slices were treated at 4°C for 24 hours with a mouse antibody against the GFAP (1:1000, MAB360, MilliporeSigma) diluted in PBS containing 0.5% BSA and 0.25% Triton X-100. Slices were then treated with a swine anti-mouse/horseradish peroxidase conjugate (Thermo Fisher Scientific) diluted 1:1000 in TNB buffer (0.1M Tris-HCl, pH 7.5; 0.15M NaCl; 0.05% Tween 20; MilliporeSigma) for 1 hour. Sections were washed 3 times in 0.01M PBS and incubated in a biosignal tyramide-fluorescein (BT-FITC) conjugate (NEN Life Sciences Products) diluted 1:100 in amplification diluent for 20 seconds at RT. Sections were washed for 3 times, for 5 minutes each time, in sterilized distilled water and then were cover-slipped with 2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO; MilliporeSigma) in glycerol (MilliporeSigma) for 1 hour at RT. After rinsing, sections were treated at 4°C for 24 hours with a mouse antibody against the Casp-1 (1:1000, AG-20B-0042, AdipoGen) diluted in PBS containing 0.5% BSA and 0.25% Triton X-100. The following steps were the same as above. For the last step, sections were stained with Hoechst (B2883, MilliporeSigma) for 10 minutes in the dark place at RT. Images were captured by confocal laser-scanning microscope (Leica).
7
Inflammasome Activation Reagents Protocol
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Tanshinone I (Tan I, HY-N0134), MCC950 (HY-12815 A), and nigericin (HY-127019) were from MedChemExpress. ATP, SiO2, Pam3CSK4, poly (I:C), poly (dA:dT), and ultrapure lipopolysaccharide (LPS) were from Invivogen. Phorbol 12-myristate 13-acetate (PMA, P8139) and DMSO (D2650) were from Sigma Aldrich. The Starfect high-efficiency transfection reagent (C101-10) was from GenStar. Antibodies were used as follows: anti-mouse IL-1β (1:1000, AF-401-NA, R&D), anti-mouse caspase-1 (1:1000, AG-20B-0042, Adipogen), anti-human cleaved IL-1β (1:2000, 12242, Cell Signaling Technology), anti-human caspase-1 (1:2000, 4199 S, Cell Signaling Technology), anti-NLRP3 (1:2000, AG-20B-0014, Adipogen), anti-ASC (1:1000, sc-22514-R, Santa Cruz), anti-Flag (1:2000, 20543-1-AP, Proteintech), and anti-GAPDH (1:5000, 60004-1-Ig, Proteintech).
8
Analyzing Caspase-1 and Caspase-11 Expression
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Cells and supernatant were lysed in RIPA buffer and sample loading buffer containing SDS and 100 mM DTT. Proteins were separated on 8–12% polyacrylamide gels. Following electrophoretic transfer of protein onto PVDF membranes (IPVH00010, Millipore), membranes were blocked in 5% skim milk and incubated with primary antibodies against caspase-1 (1:3,000 dilution, AG-20B-0042, Adipogen), caspase-11 (1:1,000 dilution, NB120-10454, Novus) and GAPDH (1:10,000 dilution, #5174, Cell Signaling Technologies). Membranes were then incubated with HRP-conjugated secondary antibody for 1 h and proteins were visualized using Super Signal Femto substrate (34096, ThermoFisher Scientific).
9
Synthesis and Characterization of Compound 8j
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Compound 8j was synthesized according to previously published protocols29 (link). VbP (Talabostat mesylate) was purchased from R&D Systems and was resuspended in DMSO containing 0.1% TFA to prevent cyclization. Bortezomib was purchased from LC laboratories, zVAD-FMK from Ubpbio, VX-765 from Apexbio Technology LLC, and etoposide from Enzo Life Sciences. LPS (from E. coli O111:B4) was purchased from Invivogen, Nigericin from Cayman Chemicals, Bestatin Methyl Ester from Santa Cruz, and MCC950 from AdipoGen. Antibodies used were: hCASP1 (no. 2225, Cell Signaling Technology), GAPDH (clone 14C10, Cell Signaling Technology), hGSDMD (NBP2-3342, Novus Biologicals), PARP (no. 9542, Cell Signaling Technology), hNLRP1 (AF6788, R&D Systems), hCARD8 (no. ab24186, Abcam), mGSDMD (no. ab209845, Abcam), GSDMD (no. 219800, Abcam), α-Actin (no. A4700, Sigma-Aldrich), mCD45 (no. 103108, Biolegend, FITC conjugate, clone 30-F11), mCD3 (no. 100235, Biolegend, APC conjugate, clone 17A2), rCD3 (no. 201411, Biolegend, PE conjugate, clone 1F4), rCD6 (no. 554904, BD Biosciences, FITC conjugate, clone OX-52), mCaspase-1 (AG20B-0042, Adipogen), mIL-1β (no. D4T2D, Cell Signaling Technologies), NLRC4 (no. ab201792, Abcam), and NLRP3 (no. ab210491, Abcam).
10
Immunoblotting for Cellular Signaling
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