Methods to detect NF-κB acetylation and methylation.
Posttranslational modifications of NF-κB, including acetylation and methylation, have emerged as an important regulatory mechanism for determining the duration and strength of NF-κB nuclear activity as well as its transcriptional output. Within the seven NF-κB family proteins, the RelA subunit of NF-κB is the most studied for its regulation by lysine acetylation and methylation. Acetylation or methylation at different lysine residues modulates distinct functions of NF-κB, including DNA-binding and transcription activity, protein stability, and its interaction with NF-κB modulators. Here, we describe the experimental methods to monitor the in vitro and in vivo acetylated or methylated forms of NF-κB. These methods include radiolabeling the acetyl or methyl groups and immunoblotting with pan- or site-specific acetyl- or methyl-lysine antibodies. Radiolabeling is useful in the initial validation of the modifications. Immunoblotting with antibodies provides a rapid and powerful approach to detect and analyze the functions of these modifications in vitro and in vivo.
Department of Biochemistry, College of Medicine, University of Illinois at Urbana-Champaign, COM 190 MSB, MC-714 506 S. Mathews Avenue, Urbana, IL, 61801, USA.
JinJing
J
Chen
Lin-Feng
LF
Chen
D000107:Acetylation; D015153:Blotting, Western; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D008745:Methylation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D051996:Transcription Factor RelA / Q000235:genetics / Q000378:metabolism
D016328:NF-kappa B; D051996:Transcription Factor RelA