Assessing acetylation of NF-kappa B

作者：Chen LF; Greene WC^*

来源：Methods, 2005, 36(4): 368-375.

DOI：10.1016/j.ymeth.2005.03.011

• RelA
• acetylation
• deacetylation
• p300
• HDAC3
• lysine

To achieve its full biological activity, NF-B-Kappa must undergo a variety of post-translational modifications, including acetylation. Acetylation plays a prominent role in regulating the nuclear action of NF-B-Kappa. The RelA subunit of NF-B-Kappa forms the major target of acetylation at several different sites. Acetylation of discrete lysine residues in RelA modulates distinct functions of NF-B-Kappa, including transcriptional activation, DNA binding, and assembly with its inhibitor IKappaB alpha. Here, we describe the experimental methods that have allowed the detection and functional analysis of acetylated forms of NF-B-Kappa. Acetylation of NF-B-Kappa can be studied both in vivo and in vitro. In vivo [3 H]acetate labeling assays provides a useful, albeit rather insensitive, method for initial verification of acetylation of either over-expressed or endogenous subunits of NF-B-Kappa. 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-B-Kappa acetylation both in vivo and in vitro.

• 出版日期2005-8