Assessing acetylation of NF-kappaB.


Journal Article

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.

Full Text

Cited Authors

  • Chen, L-F; Greene, WC

Published Date

  • August 2005

Published In

Volume / Issue

  • 36 / 4

Start / End Page

  • 368 - 375

PubMed ID

  • 16111893

Pubmed Central ID

  • 16111893

Electronic International Standard Serial Number (EISSN)

  • 1095-9130

International Standard Serial Number (ISSN)

  • 1046-2023

Digital Object Identifier (DOI)

  • 10.1016/j.ymeth.2005.03.011


  • eng