Western Blot Analysis of NF-κB

We extracted nuclear proteins to detect NF-κB p65 (Santa Cruz Biotechnology, CA, USA). Protein concentrations were determined using a bicinchoninic acid protein assay kit. After the membranes were being blocked with 5% skim milk at room temperature for 1 h, proteins were separated on a 12% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The membranes were incubated with HRP-conjugated secondary antibody at room temperature for 1 h. We visualized the signal using enhanced chemiluminescence reagents according to the manufacturer’s protocol. Antibodies to β-actin were used as internal controls for nuclear protein loading.