title: Hunting for serine 276-phosphorylated p65.
abstract: The transcription factor nuclear factor kappaB (NF-kappaB) is one of the central mediators of inflammatory gene expression. Several posttranslational modifications of NF-kappaB, regulating its transactivation ability, have been described. Especially phosphorylation of the NF-kappaB 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.
authors.affiliation: Laboratory of Eukaryotic Gene Expression (LEGEST), Department of Physiology, Ghent University, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.
authors.forename: Anneleen
authors.initials: A
authors.lastname: Spooren
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Kolmus
authors.forename: Linda
authors.initials: L
authors.lastname: Vermeulen
authors.forename: Karlien
authors.initials: K
authors.lastname: Van Wesemael
authors.forename: Guy
authors.initials: G
authors.lastname: Haegeman
authors.forename: Sarah
authors.initials: S
authors.lastname: Gerlo
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D055786:Gene Knockout Techniques; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D051996:Transcription Factor RelA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D012694:Serine