Transam transcription factor assay kits

Nuclear extracts were used to quantify DNA binding of the p65 subunit of NF-κB, AP-1 (c-fos and c-Jun) and Nrf2 using TransAM Transcription Factor Assay Kits (Active Motif, USA). After incubation, cells were scraped and nuclear and cytosolic fractions were isolated with Cell fractionation kit (Biovision, USA). Protein concentration was determined using Bicinchoninic Acid Kit for Protein Determination (Santa Cruz, CA, USA) according to manufacturer’s instructions. Briefly, 20 µg of protein per well was added to 96-well plates coated with consensus NF-κB sequence (5-GGGACTTTCC-3), consensus AP-1 sequence (5-TGAGTCA-3), and ARE consensus-binding site (5-GTCACAGTGACTCAGCAGAATCTG-3). After binding of transcription factors to target DNA, wells were washed and primary antibodies and HRP-conjugated secondary antibodies were added, respectively. The plate was washed after the secondary antibody incubation, and then chromogen was added into wells and absorbance at 450 nm was measured with ELISA reader (Biotek, USA).

The p50, p52, p65, c-Rel, and RelB DNA binding activity were measured by TransAM transcription factor assay kits (Active Motif, Carlsbad, CA, USA). Nuclear extracts were prepared in lysis buffer AM2 (Active Motif, USA). Nuclear extracts were incubated with the immobilized consensus sequence, and each subunit was detected using specific antibodies, according to the manufacturers’ instructions.