The presence of indicated proteins in lung homogenates from day 5 p.i. was assessed by western blotting using the following antibodies: anti-NLRP3 (Adipogen), anti-Caspase-1(14F468) (Santa Cruz sc-56036), anti-ASC (Adipogen), anti-IL-18 (Biovision, 5180R-10) and anti-Actin (Santa Cruz, sc-1615). Signals were detected following incubation with IRDye® Secondary Antibodies (LI-COR Biosciences, 1:10,000) for two hours at room temperature using the LI-COR Odyssey Fc Imaging System® (Table 1 ).
Anti il 18
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-IL-18 is a laboratory reagent that can be used to detect and measure the levels of interleukin-18 (IL-18) in biological samples. IL-18 is a proinflammatory cytokine that plays a role in various immune and inflammatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 1β, by Abcam (10 mentions)
Anti β actin, by Abcam (6 mentions)
Anti caspase 1, by Abcam (5 mentions)
Anti nlrp3, by Abcam (5 mentions)
Anti caspase 1, by Cell Signaling Technology (4 mentions)
Anti il 1β, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti il 6, by Abcam (4 mentions)
Anti asc, by Abcam (3 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (2 mentions)
Cell lysis kit, by Bio-Rad (2 mentions)
Anti tgf β, by Abcam (2 mentions)
Blocking buffer, by LI COR (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti il 8, by Abcam (2 mentions)
29 protocols using anti il 18
1
Lung Homogenate Protein Analysis
2
Quantitative Immune Profiling of Solid Tumors
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Collected tumors or lungs were processed as previously described 9 . We used the following antibodies: anti-CD3 and anti-CD8 (Roche, 790-4341, 750-4460 respectively), anti-IL-18 (BioVision, dil 1/200), and αPD-L1 (Dako, clone 22C3, dil 1/50), anti-Ki67 (Abcam, clone sp6, dil 1/100). After staining, slides were captured and analyzed using NDP view2 software. For the analyses, 5 zones per tumor were randomly selected and cells were counted using Figdi software. Results are expressed as number of positive cells per total cell number.
3
Western Blotting for Inflammasome Proteins
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All western blotting procedures were performed as previously described (30 (link)) with the following primary antibodies: anti-PRX3, anti-NLRP3 (Abclonal Biotechnology, Wuhan, China), anti-gasdermin D (GSDMD), anti-caspase-1, anti-cleaved caspase-1, anti-IL-1β (all from Cell Signaling Technology, Boston, MA, USA), anti-IL-18, anti-PRX5 (Abcam, Cambridge, UK), PRX6 (Zen bio., Chengdu, China) and anti-β-actin (Bimake, Houston, TX, USA).
4
Apoptosis Signaling Pathway Analysis
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Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 0.1 mg/ml pepstatin, 0.1 mg/ml leupeptin, and 0.1 mg/ml aprotinin). The protein concentration was determined by using a bicinchoninic acid protein assay. Protein lysates (40 μg) were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA), and blotted with different primary antibodies [anti-IL-18; Abcam, Cambridge, UK; anti-GSK-3β, anti-phosphorylated GSK-3β (p-GSK-3β), anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, anti-cleaved caspase-7; all from Cell Signaling Technology, Boston, MA, USA] overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with an ECL reagent (GE Healthcare, London, UK).
5
Protein Isolation and Western Blot Analysis
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Total protein was isolated from lung tissues and RAW264.7 cells using RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was measured using a BCA kit (KeyGen Biotech). An equal volume per sample was subjected to 10% SDS-PAGE and the proteins were transferred onto PVDF membranes (Millipore, Bedford, MA). The membranes were blocked in 5% non-fat milk and probed with anti-GGPPS1 (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA), anti-IL-1β (1:1000, Abcam), anti-IL-6 (1:1000, Abcam), anti-IL-18 (1:200, Abcam), anti-TNF-α (1:1000, Abcam), anti-LC3B (1:200, Abcam), anti-ATG5 (1:1000, Cell Signaling Technology, Danvers, MA), anti-Beclin1 (1:1000, Cell Signaling Technology), anti-Rab37 (1:500, Abcam), and anti-Actin (1:1000, Santa Cruz Biotechnology). After washing, the membranes were probed with HRP-conjugated secondary antibodies (1:2000, Abcam) and the signal was detected by the classical ECL method.
6
Immunoblotting for Cell Death Pathway
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Anti-GSDMD and anti-IL-18 were purchased from Abcam (Cambridge, UK). Anti-GAPDH, anti-cleaved Caspase 3, and anti-Caspase 3 were purchased from Cell Signaling Technology (Danvers, MA, US). Anti-Caspase 11 was purchased from Novus (Littleton, CO, USA). Anti-Caspase 1(p20) was purchased from AdipoGen (San Diego, CA, USA). Anti-HA was from Biolegend (San Diego, CA, USA). HT-29 cells were maintained in DMEM containing 10% (vol/vol) FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml).
7
Quercetin Modulates Neuroinflammatory Pathways
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Quercetin (98%, Figure 1 A) was purchased from Sigma‐Aldrich. Primary antibodies including Postsynapticdensity 93 (PSD93), Postsynapticdensity 95 (PSD95), Nerve growth factor (NGF), SIRT1, ASC, NLRP3 and IL‐1β were purchased from Cell Signaling Technology, Inc. Anti–brain‐derived neurotrophic factor (BDNF), anti‐NLRP3, anti–β‐actin were purchased from Abcam, Inc. Anti–IL‐18 and anti–cysteinyl‐aspartate‐specific proteinase‐1 (Cleaved Caspase‐1) were purchased from Affinity Biosciences. Secondary antibodies (antimouse IgG and anti‐rabbit IgG) were also from CST, Inc.
8
Western Blot Analysis of Intestinal Proteins
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The protein expression in intestine tissue (n = 6) was detected by Western blot as previously mentioned [20 (link)]. Briefly, RIPA lysis buffer was used to extract the total protein and nucleoprotein were extracted with RIPA lysis at 14000g for 15 min. The protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after determining the protein concentration. Then the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes. The resulting blots were blocked with 8% skim milk and incubated with anti-occludin antibody (1:1000; Proteintech, China), anti-ZO-1 antibody (1:1000; Proteintech, China), anti-TXNIP antibody (1:1000; Proteintech, China), anti-NLRP3 antibody (1:1000; Cell Signaling Technology, USA), anti-Caspase1 antibody (1:1000; Proteintech, China),anti-IL-1β antibody (1:1000; Abcam, USA), anti-IL-18 (1:1000; Abcam, USA),anti-N-terminal of GSDMD(1:1000; Abcam, USA), anti-β-actin antibody (1:10000; Santa Cruz Biotechnology, USA) overnight at 4 °C. Then, the bolts were incubated with horseradish peroxidase-conjugated secondary antibodies (1: 10000; Abcam, USA) for 1 h at 37 °C. The proteins were detected with the chemiluminescence (ECL) system. The expressions of proteins were normalized to β-actin as a reference.
9
Western Blot Analysis of Inflammatory Markers
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Protein was extracted from 50-mg samples with a cell lysis kit (Bio-Rad Laboratories, Hercules, CA, USA). The samples were incubated in buffer (246 μL lysis buffer, 1.25 μL phosphatase inhibitor, 0.25 μL protease inhibitor, and 2.5 μL PMSF) on ice for 10 min and then centrifuged. For Western blotting, equal amounts of protein supernatant (60 μg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes for immunoblotting. The membranes were blocked with blocking buffer (Li-cor, Lincoln, NE, USA) for 2 h and then incubated with either anti-CKLF-1 (1:100, Peking University Center for Human Disease Genomics, Peking University, Beijing, China), anti-IL-6 (1:500, Abcam, Cambridge, UK), anti-IL-8 (1:500, Abcam), anti-IL-18 (1:500, Abcam), anti-TGF-β (1:500, Abcam), or anti-β-actin (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies for 12 h at 4 °C. The membranes were incubated with secondary antibodies (Li-cor, Lincoln, NE) at a 1:10,000 dilution in the dark for 1 h at room temperature and then detected with a double colour infrared laser imaging system (Odyssey, Li-cor, Lincoln, NE, USA).
10
Inflammatory Cytokine Profiling in DSS-Induced Colitis
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Dextran sodium sulfate (DSS) was obtained from MP Biomedicals (MW; 36000–50000, MP Biomedicals, USA). Absolute ethanol and xylene (Chemical Reagent, Sinopharm Group, China) were purchased. Enzyme-linked immunosorbent assay (ELISA) kits for IL-6 (#CSB-E04639m), IL-1β (#CSB-E08054m), IL-18 (#CSB-E04609m), IL-17 (#CSB-E04608m), TNF-α (#CSB-E04741m), and IL-10 (#CSBE04594m) were purchased from Cusabio Biotech Co. (Wuhan, Hubei, China). Anti-NLRP3 (#AG-20B-0014-C100, Adipogen, USA), Anti-ASC (#SC-22514R, Santa Cruz Biotechnology, USA), anticaspase 1 p10 (#SC-22166, Santa Cruz Biotechnology, USA), anti-IL-1β (#12242, Cell Signaling, USA), anti-IL-18 (#ab68435, Abcam, USA), anti-ZO-1 (#sc-33725; Santa Cruz Biotechnology, USA), antioccludin-1 (#71–1500, Invitrogen, USA), anticlaudin-1 (#51–9000, Invitrogen, USA), and anti-β-actin (#ab6276,Abcam, USA) antibodies were used. TRIzol reagent (Life Company, USA), DEPC water (Biyuntian Biotechnology, China), isopropanol (Chengdu Kelon Chemical, China), SYBR Premix Ex TaqTM, PrimeScriptTM RT master mix (#RR820A; Takara Bio, Inc), the hematoxylin-eosin staining kit (Cusabio Biotech, China), the DAB color reagent histochemistry kit (Cusabio Biotech, China), the BCA protein quantitative detection kit (#G2026, Cusabio Biotech, China), ECL (#G2014, Cusabio Biotech, China), and TBS buffer (#G0001-2L, Cusabio Biotech, China) were purchased.
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