Western Blot Analysis of Protein Expression

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)].