Hunting for Serine 276-Phosphorylated p65

Spooren, Anneleen; Kolmus, Krzysztof; Vermeulen, Linda; Wesemael, Karlien Van; Haegeman, Guy; Gerlo, Sarah

doi:10.1155/2010/275892

Cited by 14 publications

(13 citation statements)

References 13 publications

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“…A study has already been published showing the lack of specificity of this and other phospho-ser276-p65 antibodies in a number of cell lines [14]. Our findings extend the list of targets, namely primary neurons and brain cells, against which the 3037 antibody does not recognize Ser276-phosphorylated p65.…”

Section: Discussionsupporting

confidence: 50%

Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies

Herkenham

Rathore

Brown

et al. 2011

Journal of Neuroinflammation

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BackgroundThe characterization and cellular localization of transcription factors like NF-κB requires the use of antibodies for western blots and immunohistochemistry. However, if target protein levels are low and the antibodies not well characterized, false positive data can result. In studies of NF-κB activity in the CNS, antibodies detecting NF-κB proteins have been used to support the finding that NF-κB is constitutively active in neurons, and activity levels are further increased by neurotoxic treatments, glutamate stimulation, or elevated synaptic activity. The specificity of the antibodies used was analyzed in this study.MethodsSelectivity and nonselectivity of commonly used commercial and non-commercial p50 and p65 antibodies were demonstrated in western blot assays conducted in tissues from mutant gene knockout mice lacking the target proteins.ResultsA few antibodies for p50 and p65 each mark a single band at the appropriate molecular weight in gels containing proteins from wildtype tissue, and this band is absent in proteins from knockout tissues. Several antibodies mark proteins that are present in knockout tissues, indicating that they are nonspecific. These include antibodies raised against the peptide sequence containing the nuclear localization signals of p65 (MAB3026; Chemicon) and p50 (sc-114; Santa Cruz). Some antibodies that recognize target proteins at the correct molecular weight still fail in western blot analysis because they also mark additional proteins and inconsistently so. We show that the criterion for validation by use of blocking peptides can still fail the test of specificity, as demonstrated for several antibodies raised against p65 phosphorylated at serine 276. Finally, even antibodies that show specificity in western blots produce nonspecific neuronal staining by immunohistochemistry.ConclusionsWe note that many of the findings in the literature about neuronal NF-κB are based on data garnered with antibodies that are not selective for the NF-κB subunit proteins p65 and p50. The data urge caution in interpreting studies of neuronal NF-κB activity in the brain.

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Section: Discussionsupporting

confidence: 50%

Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies

Herkenham

Rathore

Brown

et al. 2011

Journal of Neuroinflammation

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“…4B, in vitro phosphorylation of the RHD of c-Rel and the S267A mutant did not induce any significant change of the reactivity with the phosphospecific antibody. This differs from the massive phosphorylation that has been reported when RelA/p65 was used as a substrate in in vitro kinase assays using the same Ab for immunoblotting (39). To confirm this issue, radioactive phosphate incorporation into the recombinant RHD of c-Rel was assessed.…”

Section: Production Of Il-23 By Different Stimuli-zymosan and Lpsmentioning

confidence: 83%

Notch- and Transducin-like Enhancer of Split (TLE)-dependent Histone Deacetylation Explain Interleukin 12 (IL-12) p70 Inhibition by Zymosan

Álvarez

Municio

Hugo

et al. 2011

Journal of Biological Chemistry

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IL-12 p70 (1) and IL-23 (2) are cytokines released by antigenpresenting cells that are involved in the induction or amplification of the T-helper type 1 and type 17 responses, respectively. IL-12 p70 and IL-23 share a common chain IL-12 p40 (il12/23b) and differ by another chain, IL-12 p35 (il12a) in the case of IL-12 p70 and IL-12 p19 (il23a) in IL-23. The production of IL-12 p70 and IL-23 is regulated at the transcriptional level and determined by their heterodimeric structure. il12/23b regulation depends on NF-B activation (3), whereas the transcriptional regulation of il12a also requires a type I interferon autocrine-paracrine loop involving interferon regulatory factors (IRF)-1, IRF-3, and IRF-8/ICSBP (3-6). Stimulation of Toll-like receptor (TLR) 3 -4 induces an IL-12/IL-23 balance different from that elicited through the TLR2 and C-type lectin routes (7,8). At first glance, this may be explained because, unlike TLR2, TLR4 ligands activate the MyD88 and the TIR domain-containing adapter-inducing interferon-␤ routes and trigger both the NF-B pathway and the type I IFN autocrine-paracrine loop. However, this does not explain why TLR2 ligands may behave as negative modulators of IL-12 p70 production (7, 9) nor the molecular mechanisms underlying the distinct patterns of IL-23/IL-12 p70 response. Whereas LPS induces both cytokines, Mycobacterium tuberculosis (7) and zymosan, an extract of the cell wall of Saccharomyces cerevisiae mainly composed of ␤-glucans that is recognized by at least the C-type lectin receptor dectin-1 (10) and TLR2 (11, 12), induce IL-23 and a low amount of . Moreover, coligation of the ␤-glucan receptor dectin-1 and TLR2 enhances IL-23 and down-regulates IL-12 p70, but there is no mechanistic explanation for this finding (8,16). Addressing whether the effect of zymosan occurs via inhibition of il12a transcription or through a sole stimulation of il23a transactivation has pathophysiological relevance, because IL-23 takes part in the defense against pathogens and in the development of autoimmunity. Several hypotheses can be put forward to explain the distinct IL-12/ IL-23 balances elicited by different stimuli as follows. (i) il23a induction could be explained through the activation of transcription factors of the family ATF-2/CREB. In fact, competition between CRE-binding protein (CREB) and NF-B for the coactivator CREB-binding protein (CBP) explains the IL-12 p70 ϩ/Ϫ /IL-10 ϩϩϩ pattern induced by zymosan in dendritic cells (DC) (17). A corollary to this finding is that stimuli with a

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“…To ensure the antibody specificity for detection of phospho-Ser 276 RelA (37), WCE from control or TNF stimulated RelA +/+ and RelA −/− MEFs were prepared and assayed by western blot. We noted a significant induction of phospho-Ser 276 RelA band in ATM +/+ MEFs while no signal was detected in ATM −/− MEFs.…”

Section: Resultsmentioning

confidence: 99%

ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

et al. 2014

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Ataxia-telangiectasia mutated (ATM), a member of the phosphatidylinositol 3 kinase-like kinase family, is a master regulator of the double strand DNA break-repair pathway after genotoxic stress. Here, we found ATM serves as an essential regulator of TNF-induced NF-kB pathway. We observed that TNF exposure of cells rapidly induced DNA double strand breaks and activates ATM. TNF-induced ROS promote nuclear IKKγ association with ubiquitin and its complex formation with ATM for nuclear export. Activated cytoplasmic ATM is involved in the selective recruitment of the E3-ubiquitin ligase β-TrCP to phospho-IκBα proteosomal degradation. Importantly, ATM binds and activates the catalytic subunit of protein kinase A (PKAc), ribosmal S6 kinase that controls RelA Ser 276 phosphorylation. In ATM knockdown cells, TNF-induced RelA Ser 276 phosphorylation is significantly decreased. We further observed decreased binding and recruitment of the transcriptional elongation complex containing cyclin dependent kinase-9 (CDK9; a kinase necessary for triggering transcriptional elongation) to promoters of NF-κB-dependent immediate-early cytokine genes, in ATM knockdown cells. We conclude that ATM is a nuclear damage-response signal modulator of TNF-induced NF-κB activation that plays a key scaffolding role in IκBα degradation and RelA Ser 276 phosphorylation. Our study provides a mechanistic explanation of decreased innate immune response associated with A-T mutation.

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Hunting for Serine 276-Phosphorylated p65

Cited by 14 publications

References 13 publications

Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies

Cautionary notes on the use of NF-κB p65 and p50 antibodies for CNS studies

Notch- and Transducin-like Enhancer of Split (TLE)-dependent Histone Deacetylation Explain Interleukin 12 (IL-12) p70 Inhibition by Zymosan

ATM regulates NF-κB-dependent immediate-early genes via RelA Ser 276 phosphorylation coupled to CDK9 promoter recruitment

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