NF-κB p65 DNA-binding activity assay

HUVECs in 6-well plates (3 × 10⁵/well) were exposed to TP at the indicated concentrations for 1 hour, followed by incubation with ox-LDL (50 µg/mL) for an additional 30 minutes. After incubation, nuclear protein was extracted from the cells using a nuclear extraction kit (Active Motif, Carlsbad, California), as described by the manufacturer. A specific TransAM NF-κB p65 Transcription Factor Assay Kit (Active Motif) was used to quantify the DNA-binding activity of NF-κB p65 following the manufacturer's instructions. Briefly, extracted nuclear proteins were added to each well coated with an unlabeled oligonucleotide containing the consensus binding site for NF-κB (5′-GGGACTTTCC-3′) and incubated for 1 hour. After washing, a primary antibody directed against the NF-κB p65 subunit was applied and incubated for 1 hour. Subsequently, a secondary antibody conjugated to horseradish peroxidase was added and incubated for 1 hour. A colorimetric reaction was initiated by adding a developing solution. After termination with a stop solution, the plate was read at 450 nm with an absorbance microplate reader (Molecular Devices, Downingtown, Pennsylvania). The protein levels of the nuclear extract were assayed using a protein assay kit and used to normalize the DNA-binding activity of NF-κB p65.