Selective recognition of the activated form of transcription factor NF-kappa B by a monoclonal antibody.
Transcription factor NF-kappa B is a central regulator of inflammatory, immune and acute phase reactions. It rapidly initiates the transcription of a wide variety of target genes in response to various pathogenic stimuli. Because NF-kappa B is predominantly controlled at a posttranscriptional level through association with the inhibitory I kappa B subunits, its activation cannot be monitored directly at a cellular level by means of detecting new mRNA or protein synthesis. In this study, we describe a monoclonal antibody, designated alpha-p65MAb, that recognizes an epitope which includes the nuclear location signal (NLS) of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-kappa B. alpha-p65MAb recognized human and rodent p65 only when I kappa B alpha was not bound to p65. Thus, the IgG3 selectively stained the activated, nuclear form of NF-kappa B in cultured cells. Unlike I kappa B, the MAb and its Fab fragments did not inhibit the DNA binding activity of NF-kappa B in mobility shift assays. We show that alpha-p65MAb is suitable to study the activation state of NF-kappa B in cryosections of tissues.
Kaltschmidt
C
C
Institute of Biochemistry, University of Freiburg, Germany.
Kaltschmidt
B
B
Henkel
T
T
Stockinger
H
H
Baeuerle
P A
PA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000378:metabolism; D011485:Protein Binding; D051996:Transcription Factor RelA
D000911:Antibodies, Monoclonal; D016328:NF-kappa B; D051996:Transcription Factor RelA