Activation of Nuclear Factor-B

Nuclear factor-κB (NF-κB) is a key transcription factor that regulates the expression of genes involved in immune and inflammatory responses and in cell death and survival. This chapter describes in detail the method for measuring the NF-κB binding activity in the cultured human mast cell line HMC-1 using the electrophoretic mobility shift assay: the activation of NF-κB was illustrated by adding lipopolysaccharide to mast cells, nuclear proteins were isolated, and the NF-κB binding activity was determined. We also demonstrate the specificity of the NF-κB binding activity using a competition and supershift assay with specific antibodies that recognize NF-κB subunits p50 and p65. Lipopolysaccharide caused a rapid and significant increase in NF-κB binding activity.