Ecl working solution

Total proteins of macrophages and artery were extracted by RIPA (Beyotime Biotechnology, Shanghai, China) containing 1 mM PMSF (Beyotime Biotechnology, Shanghai, China). The BCA kit (Beyotime Biotechnology, Shanghai, China) is used to determine the protein concentration. The samples were added to the sample wells of 10% SDS-PAGE gels for electrophoresis. The proteins are transferred to the PVDF membrane and then sealed with 5% skim milk for 1 h. The PVDF membrane was placed into the primary antibody at 4 °C overnight. TBS-T wash three times. The PVDF membrane was placed into the second antibody for 1h at room temperature. TBS-T wash three times. The protein bands were treated with ECL working solution (Solarbio, Beijing, China) and visualized using a gel imaging system (Tanon, Shanghai, China). The relative expression levels of the target proteins were determined with Image J software.

Total protein was extracted from macrophages or mice blood vessels using RIPA lysis buffer supplemented with 1 mM PMSF (Beyotime, China). The protein concentration was measured by BCA Protein Assay Kit (Beyotime, China). Western blot gel was established using Epizyme PAGE Gel Fast Preparation Kit. The protein was separated by SDS polyacrylamide gel electrophoresis, and transferred onto PVDF membranes (Millipore, China). Subsequently, membranes were blocked with 5% skim milk for 1 h at room temperature, and then incubated with different antibodies at 4 °C overnight. After washing with TBST three times, membranes were incubated with secondary antibody for 1 h at room temperature, including antibodies against NLRP3, Caspase-1, cleaved Caspase-1, cleaved GSDMD, IL-1β (CST, USA, 1:1000), β-actin (Affinity, USA, 1:10,000) and so on. Western blot bands were examined and analyzed by Image Lab™ Software (Bio-Rad). The protein bands were treated with ECL working solution (Solarbio, China) and visualized using a gel imaging system (Tanon, China). The relative expression levels of the target proteins were determined with Image J software.