*2.7. AP-1 and NF-*κ*B Binding Assay*

DNA-binding activities of AP-1 and NF-κB in nuclear extracts were assayed by electrophoretic mobility shift assay (EMSA) with biotin-labeled double-stranded AP-1 or NF-κB oligonucleotides (MDBio Inc., Taipei, Taiwan), as described by Chiu et al. [18]. EMSA was carried out by using the Lightshift kit from Pierce (Rockford, IL, USA). Binding reactions containing 10 μg of nuclear extracts, 1 μg poly (dI·dC), 12.5 μg poly-l-lysine, 2 pmol of oligonucleotide probe, and 2 μL of 10× binding bu ffer were incubated for 20 min at room temperature. Protein-DNA complexes were separated by electrophoresis on a 6% non-denaturing acrylamide gel, transferred to positively charged nylon membranes (Millipore, Bedford, MA, USA), and then UV cross-linked. Gel shifts were visualized with a streptavidin-horseradish peroxidase followed using chemiluminescent detection.
