After the designated treatments were performed, liver tissues and cell pellets were lysed with RIPA buffer supplemented with protease inhibitors. Total proteins were separated via 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight with rabbit antibodies against NLRP3 (Zymed, South San Francisco, CA, USA), cleaved caspase 1, cleaved caspase 3 (BD PharMingen, San Diego, California, USA), IL-1β (R&D Systems, USA), IL-18 (Santa Cruz Biotechnology Inc., Santa Cruz,California, USA), LC3-I, LC3-II (Sigma, USA), NF-κB, Beclin-1, PINK1, Parkin and β-actin primary antibodies (Cell Signaling Technology, Danvers, MA, USA) at 4°C. Then, the membranes were treated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Cell Signaling, CA, USA) and developed with a chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA). Densitometry analysis was performed using ImageJ software, and the relative levels of protein in each group were normalized to the loading control.
Nlrp3
Manufactured by Zymo Research
Sourced in United States
The NLRP3 is a laboratory equipment product designed for the detection and analysis of the NLRP3 inflammasome. The NLRP3 inflammasome is a multiprotein complex involved in the innate immune response. The product provides tools for researchers to study the structure, function, and regulation of the NLRP3 inflammasome in various biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Il 18, by Santa Cruz Biotechnology (1 mentions)
Pink1, by Cell Signaling Technology (1 mentions)
Parkin, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Cleaved caspase 3, by BD (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl solution, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
2 protocols using nlrp3
1
Western Blot Analysis of Liver Proteins
2
Western Blot Analysis of Protein Expression
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After isolating the total proteins from the treated HrGECs, a bicinchoninic acid (BCA) protein assay kit (Merck, New Jersey, USA) was utilized to determine the concentration of proteins and about 40 μg proteins were loaded, followed by being separated with the 12% SDS-PAGE. Then, the proteins in the SDS-PAGE were transferred to the PVDF membrane (Merck, New Jersey, USA) and the membrane was subsequently incubated with 5% BSA for 2 hours, followed by being incubated with the primary antibody against NADPH oxidase-2 (NOX-2) (1:1200, Zymed, California, USA), NLRP3 (1:1500, Zymed, California, USA), apoptosis-associated speck-like protein containing a CARD (ASC) (1:800, Zymed, California, USA), p-AMPKα (1:500, Zymed, California, USA), AMPKα (1:1600, Zymed, California, USA), mTOR (1:1500, Zymed, California, USA), and β-actin (1:8000, Zymed, California, USA) overnight. The membrane was exposed to enhanced chemiluminescence (ECL) solution (Invitrogen, California, USA) after 1.5 hours of incubation in secondary antibody solution (1:2000, Zymed, California, USA) and the relative expression level of target proteins was confirmed by visualization with Image J software [16 (link)].
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