Assessing acetylation of NF-kappaB

• Published in: Methods (San Diego, Calif.) Vol. 36; no. 4; pp. 368 - 375
• Main Authors: Chen, Lin-Feng, Greene, Warner C
• Format: Journal Article
• Language: English
• Published: United States 01.08.2005
• Subjects: Acetates - immunology; Acetates - metabolism; Acetylation; Acetyltransferases - immunology; Acetyltransferases - metabolism; Animals; Antibodies - immunology; Cell Cycle Proteins - immunology; Cell Cycle Proteins - metabolism; Cell Extracts - isolation & purification; Cell Line; Clinical Laboratory Techniques; Histone Acetyltransferases; Humans; Immunoprecipitation - methods; Lysine - immunology; Lysine - metabolism; NF-kappa B - metabolism; p300-CBP Transcription Factors; Protein Processing, Post-Translational; Radioisotopes - chemistry; Transcription Factor RelA; Transcription Factors - immunology; Transcription Factors - metabolism; Transfection - methods
• ISSN: 1046-2023

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.