[17] Redox regulation of activation of NF-κB transcription factor complex: Effects of N-acetylcysteine

Publisher Summary This chapter describes detailed methods on the appropriate cell culture conditions to study the influence of oxidants and antioxidants, the preparation of nuclear protein extracts, and the use of gel retardation assays to detect the presence of activated NF- K B in the nucleus. The chapter also describes a reporter gene-based system to assay whether the DNA-bound NF- K B is transcriptionally active. NF- K B is a heterodimeric transcription factor complex composed, in its classic form, of two DNA-binding subunits: p50 and p65. NF- K B was first identified as a DNA-binding activity specific for the KB motif in the immunoglobin kappa ( K ) enhancer. Cloning of the NF- K B p50 and p65 genes revealed a family of NF- K B/rel proteins that participate in a variety of transcriptionally regulated processes, such as lymphocyte differentiation, responsiveness to cytokines, and embryonic development in Drosophila. NF- K B is present in many cell types in an inactive form in the cytoplasm, bound to a cytoplasmic retention molecule called I- K B (for inhibitor of NF- K B). Nuclear translocation of NF- K B is achieved after stimulation of the cells by many different inducers (cytokines, phorbol esters, viral proteins, oxidants) and presumably involves phosphorylation of I- K B which disrupts its interaction with the p65 NF- K B subunit. The NF- K B complex is then free to migrate to the nucleus and transactivate its various target genes.