Activation of nuclear factor-kappaB.
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.
Li
Chuanfu
C
Department of Surgery, Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA.
Kelley
Jim
J
Ha
Tuanzhu
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D005786:Gene Expression Regulation; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008407:Mast Cells / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism
D008070:Lipopolysaccharides; D016328:NF-kappa B; D009687:Nuclear Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate