2.10. NF-κB DNA-Binding Activity by Electrophoretic Mobility Shift Assay (EMSA)

SK-N-SH cells were infected with HSV-1 (MOI = 0.01) for 2 h and then treated with QA at concentrations of 50 and 100 μg/mL for 48 h. A DIG Gel Shift kit (Roche) was used for the EMSA. An NF-κB oligonucleotide probe (5′-CTT GAA GGG ATT TCC CTG GCT TGA AGG GAT TTC CCT GG-3′ (only sense strands are shown; consensus sequences for NF-κB are underlined)) containing the NF-κB binding motif was end-labeled with DIG-ddUTP. For the binding reaction, 10 μg of sample protein was incubated at room temperature for 30 min with the DIG-labeled probe. The DNA-protein complexes were separated by electrophoresis in 6% nondenaturing polyacrylamide gels using 0.5× TBE as a running buffer. After electrophoresis, the gels were transferred to nylon membranes and detected by chemiluminescence. Signal intensity was quantified with an image analyzer.