2.4. Western Blot Analysis

The expression of inflammatory proteins was analyzed using the western blot assay. Cells were seeded into plates, treated, and incubated to the desired time point. The treatment times for the different biological processes are shown in Scheme 1. The treated cells were then washed, collected, and lysed with the RIPA lysis buffer containing protease and phosphatase inhibitors. Protein estimation was performed using the Bradford assay, and 30 μg of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein bands in the gel were transferred to nitrocellulose membranes and incubated overnight with primary antibodies against α-tubulin, iNOS, COX-2, ERK, pERK, JNK, pJNK, p38, p-p38, NF-κB, histone-H3, β-actin, IκB, pIκB, c-Fos, pc-Fos, c-Jun, or pc-Jun. On the next day, the membranes were washed and then incubated with the respective secondary antibodies, and the protein bands were finally visualized via the Chemiluminesent ECL Western Blotting Detection Reagent (Amersham Pharmacia Biotech, Little Chalfont, UK). A quantitative evaluation of the bands was conducted using the Image Master™ 2D Elite software (version 3.1, Amersham Pharmacia Biotech, Little Chalfont, UK).