J774A.1 cells were seeded on 6-well plates (2 × 106 cells/well) in RPMI 1640 medium for 24 h. Cells were then primed and activated with LPS/ATP and treated with JC-171 as described above. After treatment, cells were collected and wash with ice-cold PBS twice. Cell pellets were lysed by sonication in a radioimmunoprecipitation (RIPA) buffer solution. Protein samples were quantified by the Bradford method. Equal amounts of protein (20.0 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Proteins were probed with primary antibodies overnight at 4 °C: anti-NLRP3 (1:800, abcam, U.S.A.), anti-IL1β (1:500, EMD Millipore, CA, U.S.A.), anticaspase-1 (P20) (1:1000, Cell Signaling Technology, MA, U.S.A.), anticaspase-1 (P10) (1:200, Santa Cruz Biotechnology, Santa Cruz, U.S.A.) and incubated with a 1:1000 dilution of horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Cell Signaling Technology, U.S.A.). After the proteins were washed three times in TBS-Tween 20 for 10 min, they were visualized by employing chemiluminescent reagent (Thermo Fischer Scientific, Waltham, MA). The blots were also probed with antibodies against α-tubulin to ensure equal loading of proteins.
Anti il 1β
Manufactured by Merck Group
Sourced in United States
Anti-IL-1β is a laboratory reagent used for the detection and measurement of the cytokine interleukin-1 beta (IL-1β) in various biological samples. It is a highly specific and sensitive tool for researchers studying inflammatory processes and related diseases.
Automatically generated - may contain errors
Lab products found in correlation
Anti nlrp3, by Abcam (2 mentions)
Chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Anti caspase 1 p10, by Santa Cruz Biotechnology (1 mentions)
Nlrp3 clone d4d8t, by Cell Signaling Technology (1 mentions)
Igg1 rtk2071, by BioLegend (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Human ifn γ, by Thermo Fisher Scientific (1 mentions)
Murine ifn γ, by Thermo Fisher Scientific (1 mentions)
Palmitate bsa, by Agilent Technologies (1 mentions)
M csf, by Thermo Fisher Scientific (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Oligomycin, by Merck Group (1 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Merck Group (1 mentions)
Antimycin a, by Merck Group (1 mentions)
Etomoxir, by Merck Group (1 mentions)
Murine il 4, by Thermo Fisher Scientific (1 mentions)
Mitotempo, by Merck Group (1 mentions)
Anti pmp70, by Abcam (1 mentions)
Anti catalase, by Santa Cruz Biotechnology (1 mentions)
8 protocols using anti il 1β
1
NLRP3 Inflammasome Activation Assay
2
Flow Cytometry Analysis of Immune Cells
3
Immunoblotting Antibody Panel Validation
4
Oxidized CaMKII Regulation by IL-1β
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Left ventricle tissue samples were incubated for 24 h in the presence or absence of 10 ng ml−1 IL-1β and subsequently homogenized. Protein was measured by the Bradford method using BSA as standard. Lysates (∼90 μg of total protein per gel line) were seeded in a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Blots were probed overnight with antibodies raised against oxidized CaMKII (referred as Ox-CaMKII), Cat# 07-1387, 1:1,000 (Millipore Corp, USA). After stripping the blots were probed with phospho-Thr286-CaMKII (referred as p-CaMKII), Cat# 32678 1:1,000 (Badrilla, UK), Anti IL-1β Cat# I3767 1:1,000 (Sigma, USA) and Anti GAPDH, Cat# MAB374, 1:2,000 (Santa Cruz Biotechnology, USA) was used for normalization. Immunoreactivity was visualized by a peroxidase-based chemiluminescence detection kit (Amersham Biosciences) using a Chemidoc Imaging System. The signal intensity of the bands in the immunoblots was quantified by densitometry using the Image J software (NIH, USA).
5
Substantia Nigra Protein Extraction and Analysis
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The substantia nigra tissue was cut it into small pieces, and then 400 µL of single detergent lysis solution (including PMSF) was added. The solution was lysed for 30 min and centrifuged at 12,000 rpm at 4 °C for 5 min, and the supernatant was then collected. The concentration of the protein to be tested was measured and recorded and SDS–PAGE electrophoresis (voltage 40 V) was performed. The proteins were transferred to a membrane, which was stained with 1× Ponceau staining solution for 5 min, moved to 5% skim milk, sealed for 2 h, incubated with the primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Cell Signaling Technology, Danvers, MA) for 2 h at room temperature, and finally subjected to chemiluminescence, development, and fixation. Following primary antibodies were used: anti-TH (1:1000; Sigma, T2928), anti-NLRP3 (1: 1000, BioVision, A1767-100), anti-IL-1β (1:1000; Sigma, I-3767); anti-Capase-1 (1:1000; Sino Biological, 90,011-MM02) anti-GAPDH (1:1000, Sino Biological, MB10094-T52).
6
Cytokine and Antibody Procurement
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Human recombinant IL-20 protein, anti-IL-20 monoclonal antibody, and isotype IgG1 antibody were purchased from R&D Systems (Minneapolis, MN, USA), and anti-IL-1β and anti-bone morphogenetic protein-2 (BMP-2) monoclonal antibodies were purchased from Sigma (St. Louis, MO USA). Recombinant human IL-1β and BMP-2 were purchased from PeproTech (Rocky Hill, NJ, USA).
7
Western Blot Analysis of Protein Markers
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The protein lysates were prepared with protease inhibitors cocktail‐contained radioimmunoprecipitation assay lysate (Beyotime). The protein samples were boiled at 98°C for 10 min. Next, 60 μg of proteins was used for analysis on 10% sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (SDS‐PAGE; Bio‐Rad, Hercules, CA). The SDS‐PAGE gels were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Bio‐Rad) for 45 min. Then, the protein‐carried PVDF membranes were blocked with 2% bovine serum albumin, and they were incubated with the indicated primary antibody overnight. Afterward, the membranes were incubated with horseradish peroxidase‐conjugated secondary antibody for 45 min diluted at 1:50,000 (Bioworld Technology, Nanjing, China). Importantly, the primary antibodies including anti‐GAPDH, anti‐FBXW7, anti‐Bax, anti‐Bcl‐2, anti‐TNF‐α, anti‐IL‐1β, anti‐IL‐6, anti‐CAT, anti‐GSH, and anti‐SOD1 were purchased from Sigma‐Aldrich.
8
Inflammatory Signaling Pathway Modulation
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The sfd-FLS line HIG-82 (American Type Culture Collection cat. no. 1832) was purchased from BeNa Culture Collection. sfd-FLSs were grown in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO2. Cells were treated with IL-1β (1 mg/ml; cat no. SRP6551; Sigma-Aldrich; Merck KGaA), anti-IL-1β (1 mg/ml; cat no. PRS4877; Sigma-Aldrich; Merck KGaA) and/or NF-κB inhibitor (1 mg/ml; cat no. 481412; Sigma-Aldrich; Merck KGaA) for 12 h at 37°C for further analysis.
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