Nfκb p65 ez tfa transcription factor assay colorimetric kit

We used the nuclear extraction kit (Sigma-Aldrich) to extract proteins in the nucleus according to the manufacturer’s manual. For the activity assay of NFκBp65, we employed the NFκB p65 EZ-TFA Transcription Factor Assay Colorimetric Kit purchased form Millipore (Temecula, CA, USA) according to the instruction manual.

Cell samples were washed and collected. The pellet was added to hypotonic buffer (10 mmol/L HEPES, 1 mmol/L MgCl₂, 10 mmol/L KCl, 0.5 mmol/L DTT, 1 mmol/L EDTA, 20 μg/mL aprotinin, 4 μg/mL leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40), and stood on ice for 20 min. Afterwards, crude nuclei were acquired after centrifugation at 6000× g for 20 min and pellets were resuspended in hypertonic buffer (10 mmol/L HEPES, 1 mmol/L MgCl₂, 400 mmol/L KCl, 0.5 mmol/L DTT, 1 mmol/L EDTA, 20 μg/mL aprotinin, 4 μg/mL leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 25% glycerol). After incubating for an additional 30 min, the nuclear extracts were obtained by centrifugation at 10,000× g for 20 min and were frozen at −80 °C. In the NFκB p65 assay, we used NFκB p65 EZ-TFA Transcription Factor Assay Colorimetric Kit (Millipore, Temecula, CA, USA). Briefly, the NFκB response element (a double stranded DNA sequence) was immobilized onto the bottom of the wells of a 96-well plate. NFκB of nuclear extract bound to the NFκB response element, and was identified using an anti NFκB p65 antibody. A secondary antibody conjugated to HRP was added and then it reacted with the substrate to provide a colorimetric readout at 450 nm.