Assessing acetylation of NF-κB

To achieve its full biological activity, NF-κB must undergo a variety of post-translational modifications, including acetylation. Acetylation plays a prominent role in regulating the nuclear action of NF-κB. The RelA subunit of NF-κB forms the major target of acetylation at several different sites. Acetylation of discrete lysine residues in RelA modulates distinct functions of NF-κB, including transcriptional activation, DNA binding, and assembly with its inhibitor IκBα. Here, we describe the experimental methods that have allowed the detection and functional analysis of acetylated forms of NF-κB. Acetylation of NF-κB 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-κB. 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-κB acetylation both in vivo and in vitro.