Measurement of NF-κB DNA binding activity

DNA binding activity of NF-κB was measured with a sensitive multiwell colorimetric assay³⁷ using a TransAM-NFkappaB kit (Active Motif; Carlsbad, CA, USA). Briefly, nuclear extracts (10 μg) from each sample were incubated in 96-well plates coated with NF-κB consensus double-stranded oligonucleotide sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) for 1 h and then with primary NF-κB antibody (1:1000) for 1 h, and subsequently with peroxidase-conjugated secondary antibody (1:1000) for 1 h at room temperature. After colorimetric reaction, optical density was read at 450 nm with a microplate reader (ELx800, BioTek). The results are expressed after subtraction of the blank values. For competition assays, the nuclear extracts were incubated with 22-bp double-stranded DNA, either wild-type or mutated (5′-AGTTGAGCTCACTTTCCC AGGC-3′ [underline denotes the substitution]).