2.8. Quantification of NF‐κB (p65) DNA‐binding activity

The NF‐kB p65 DNA binding activity was measured using an ELISA‐based method through NF‐kB p65 Transcription Factor assay kit (ab133112; Abcam, UK). In brief, nuclear extracts of NIH/3T3 cells were incubated for 1 h at room temperature on a 96‐well plate pre‐coated with a double‐stranded DNA sequence containing an immobilized NF‐kB response element. NF‐kB, present in nuclear extracts, binds to the NF‐kB response element and is detected using an antibody for NF‐kB/p65 as previously described. ³⁵ A secondary antibody conjugated to HRP is then added and NF‐kB activation is measured with a plate absorbance reader at 450 nm (Thermo Scientific MULTISKAN GO, Finland).