Ma5 15160

Cells were collected and lysed to obtain proteins. Protein concentration was determined using the bicinchoninic acid method. Each lane was added with 20 μg total protein sample. Protein isolation was performed on 12% SDS-polyacrylamide gel electrophoresis at 120 V for 90 min. The isolated protein was moved to polyvinylidene fluoride membranes at 250 mA for 90 min. Then, the membranes were incubated with 50 g/L TBST buffer for 1 h and sealed and added with SIRT1 antibody (1:1,000, ab189494; Abcam), NF-κB p65 antibody (1:1,000, ab32536; Abcam), and NF-κB p-p65 antibody (1:1,000, MA5-15160; Thermo Fisher Scientific, Inc.) at 4°C overnight. The next day, after washing, the membranes were hybridized with secondary goat anti-rabbit IgG antibody labeled with anti-biotin (1:2,000, ab6721; Abcam) and incubated at 37°C for 1 h. After washing, the membranes were added with an enhanced chemiluminescence luminescent reagent. GelDoc software was used for image acquisition and data analysis. PCNA (1:1,000, ab16048; Abcam) was used as the loading control for nuclear NF-κB p65 and NF-κB p-p65, and β-actin (1 μg/mL, ab8227; Abcam) was used as the internal reference for others.