Assessing acetylation of NF-kappaB.
To achieve its full biological activity, NF-kappaB must undergo a variety of post-translational modifications, including acetylation. Acetylation plays a prominent role in regulating the nuclear action of NF-kappaB. The RelA subunit of NF-kappaB forms the major target of acetylation at several different sites. Acetylation of discrete lysine residues in RelA modulates distinct functions of NF-kappaB, including transcriptional activation, DNA binding, and assembly with its inhibitor IkappaBalpha. Here, we describe the experimental methods that have allowed the detection and functional analysis of acetylated forms of NF-kappaB. Acetylation of NF-kappaB can be studied both in vivo and in vitro. In vivo [3H]acetate labeling assays provides a useful, albeit rather insensitive, method for initial verification of acetylation of either over-expressed or endogenous subunits of NF-kappaB. A second valuable in vivo approach involves the use of anti-acetylated lysine antibodies for immunoblotting. However, the success of this approach varies with the specific antibody employed and the target protein studied. In vitro acetylation assays provide a rapid and sensitive method to validate the involvement of candidate histone acetyltransferases and to map the sites of acetylation. Anti-RelA antibodies that selectively react with site-specific acetylated forms of RelA are a singularly powerful tool for the study of NF-kappaB acetylation both in vivo and in vitro.
Chen
Lin-Feng
LF
Gladstone Institute of Virology and Immunology, University of California, San Francisco 94158, USA.
Greene
Warner C
WC
D000085:Acetates / Q000276:immunology / Q000378:metabolism; D000107:Acetylation; D000123:Acetyltransferases / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D018797:Cell Cycle Proteins / Q000276:immunology / Q000378:metabolism; D002457:Cell Extracts / Q000302:isolation & purification; D002460:Cell Line; D019411:Clinical Laboratory Techniques; D051548:Histone Acetyltransferases; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D008239:Lysine / Q000276:immunology / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011868:Radioisotopes / Q000737:chemistry; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism; D014162:Transfection / Q000379:methods; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000085:Acetates; D000123:Acetyltransferases; D000906:Antibodies; D018797:Cell Cycle Proteins; D002457:Cell Extracts; D051548:Histone Acetyltransferases; D008239:Lysine; D016328:NF-kappa B; D011868:Radioisotopes; D051996:Transcription Factor RelA; D014157:Transcription Factors; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor