Lee, J.H., Koo, T.H., Hwang, B.Y. and Lee, J.J.
Notes: To investigate the effect of the compound kamebakaurin (KA) on NF-κB, an NF-κB-responsive firefly luciferase vector was transfected into HeLa, Jurkat and THP-1 cells. The Luciferase Assay System was used to assay the level of NF-κB induction after treatment of cells with various concentrations of KA. To determine if KA influenced the DNA-binding activity of NF-κB, nuclear extracts of HeLa, Jurkat and THP-1 cells were prepared after preincubation with KA and stimulation of NF-κB activity. Control nuclear extracts were prepared from unstimulated p50- or RelA-overexpressed MCF-7 cells. In addition, the wildtype and DNA-binding mutant RelA and p50 (NF-κB) His-tagged proteins were translated using the TNT® Quick Coupled Transcription/Translation System and subsequently purified. Using the Gel Shift Assay System, the NF-κB and AP1 oligos were tested for electromobility shifts with the prepared nuclear extracts or with purified wildtype and mutant proteins. Supershift studies using anti-p50 or anti-RelA antibodies were also performed. (3113)