2.6. Western blot

Cells in six‐well plates were washed with PBS and lysed with equal volumes of RIPA lysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride (PMSF) on ice and then centrifuged for 10 minutes at 300 g under 4°C. Lysates with equal amounts of protein were separated by 10% SDS‐PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membranes were blocked for 1 hour at room temperature with 5% BSA in TBS containing 0.1% Tween‐20 and then incubated overnight at 4°C with NF‐κB (Cell Signaling Technology) or β‐actin (Santa Cruz Biotechnology) antibody. The membranes were exposed to horseradish peroxidase‐labelled secondary antibodies (1:3000) for 1 hour at room temperature and detected by enhanced chemiluminescence detection systems (Amersham Imager 600, GE Healthcare Life Sciences, and ChemiDoc™ Touch Imaging System, Bio‐Rad).