NF-κB Signaling Pathway Regulation

NF-κB-dependent reporter gene plasmid, pGL4.32[luc2P/NF-κB-RE/Hygro] (abbreviated to NF-κB-LUC hereafter), containing five copies of an NF-κB response element driving transcription of a destabilised form of Firefly Luciferase was purchased from Clontech. Control luciferase reporter gene containing only a minimal promoter (minP-LUC) was described previously [33 (link)]. Expression plasmid pCMV-p65, which expresses the RelA/p65 subunit of NF-κB, was a gift from Matthew Vincenti (Dartmouth Medical School, Lebanon, NH). Plasmid pDC515:mDIA-CT expressing a constitutively active nuclear mouse mDIA was made by subcloning the F1F2+C fragment of mDIA1 from pEF-mDIA-F1F2+C plasmid (a generous gift from John Copeland and described previously [39 (link)] into the BamH1 and Sal1 sites of pDC515 (Microbix). Recombinant adenoviral vector expressing an NLS-tagged polymerisation defective mutant of β-actin (Ad:NLS-Actin_R62D) and control adenoviral vector lacking a transgene (Ad:Control) have been described previously [37 (link)]. Plasmid expressing exportin6 (XPO6) was described previously [37 (link)]. Silencer select siRNA for IPO9 and CFL1 were purchased from Invitrogen Life Technologies.