Article

Activation of nuclear factor-kappaB.

Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA.
Methods in molecular biology (Clifton, N.J.) 02/2006; 315:141-50. pp.141-50
Source: PubMed

ABSTRACT Nuclear factor-kappaB (NF-kappaB) 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-kappaB binding activity in the cultured human mast cell line HMC-1 using the electrophoretic mobility shift assay: the activation of NF-kappaB was illustrated by adding lipopolysaccharide to mast cells, nuclear proteins were isolated, and the NF-kappaB binding activity was determined. We also demonstrate the specificity of the NF-kappaB binding activity using a competition and supershift assay with specific antibodies that recognize NF-kappaB subunits p50 and p65. Lipopolysaccharide caused a rapid and significant increase in NF-kappaB binding activity.

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Keywords

cultured human mast cell line HMC-1
 
electrophoretic mobility shift assay
 
immune
 
key transcription factor
 
mast cells
 
NF-kappaB binding activity
 
Nuclear factor-kappaB
 
nuclear proteins
 
rapid
 
recognize NF-kappaB subunits p50
 
specificity
 
supershift assay
 

Chuanfu Li