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Difficulties in generating specific antibodies for immunohistochemical detection of nitrosylated tubulins.

ABSTRACT: Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated ?- and ?-tubulin isoforms in primary neurons after fixation. The approach was based on in situ replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.

SUBMITTER: Kamnev A

PROVIDER: S-EPMC3696116 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Difficulties in generating specific antibodies for immunohistochemical detection of nitrosylated tubulins.

Kamnev Anton A Muhar Matthias M Preinreich Martina M Ammer Hermann H Propst Friedrich F

PloS one 20130628 6

Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contr ...[more]

PMID: 23840827

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Project description:Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

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Difficulties in generating specific antibodies for immunohistochemical detection of nitrosylated tubulins.

Publications

Difficulties in generating specific antibodies for immunohistochemical detection of nitrosylated tubulins.

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