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Journal of Biomedicine and Biotechnology
Volume 2010, Article ID 275892, 9 pages
Methodology Report

Hunting for Serine 276-Phosphorylated p65

Laboratory of Eukaryotic Gene Expression (LEGEST), Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium

Received 13 August 2009; Revised 11 November 2009; Accepted 17 November 2009

Academic Editor: Susan A. Rotenberg

Copyright © 2010 Anneleen Spooren et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


The transcription factor nuclear factor kappaB (NF- 𝜅 B) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF- 𝜅 B, regulating its transactivation ability, have been described. Especially phosphorylation of the NF- 𝜅 B subunit p65 has been investigated in depth and several commercial phosphospecific antibodies, targeting selected p65 residues, are available. One of the p65 residues, that is subject to phosphorylation by protein kinase A (PKA) as well as by mitogen-stimulated kinase-1 (MSK-1), is the serine at position 276. Here, we have performed a detailed analysis of the performance of the most commonly used commercial anti-P-p65 Ser276 antibodies. Our findings indicate that at least three widely used anti-P-p65 Ser276 antibodies do not detect p65 in vivo via Western Blot, but instead crossreact with PKA-regulated proteins. As PKA is one of the main kinases responsible for phosphorylation of p65 at Ser276, this observation warrants cautious interpretation of data generated using the tested antibodies.