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Poor antibody validation is a challenge in biomedical research: a case study for detection of c-FLIP

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Abstract

Successful translation of findings derived from preclinical studies into effective therapies is critical in biomedical research. Lack of robustness and reproducibility of the preclinical data, due to insufficient number of repeats, inadequate cell-based and mouse models contribute to the poor success rate. Antibodies are widely used in preclinical research, notably to determine the expression of potential therapeutic targets in tissues of interest, including tumors, but also to identify disease and/or treatment response biomarkers. We sought to determine whether the current antibody characterization standards in preclinical research are sufficient to ensure reliability of the data found in peer-reviewed publications. To address this issue, we used detection of the protein c-FLIP, a major factor of resistance to apoptosis, as a proof of concept. Accurate detection of endogenous c-FLIP levels in the preclinical settings is imperative since it is considered as a potential theranostic biomarker. Several sources of c-FLIP antibodies validated by their manufacturer and recommended for western blotting were therefore rigorously tested. We found a wide divergence in immune recognition properties. While these antibodies have been used in many publications, our results show that several of them failed to detect endogenous c-FLIP protein by Western blotting. Our results suggest that antibody validation standards are inadequate, and that systematic use of genetic knockdowns and/or knockouts to establish proof of specificity is critical, even for antibodies previously used in the scientific literature. Because antibodies are fundamental tools in both preclinical and clinical research, ensuring their specificity is crucial.

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Abbreviations

TRAIL:

TNF-related apoptosis inducing ligand

DISC:

Death-inducing signaling complex

TNF:

Tumor necrosis factor

c-FLIP:

Cellular FLICE-inhibitory protein

MAPK:

Mitogen-activated protein kinases

ERK:

Extracellular signal-regulated kinase

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Acknowledgments

Octavian Bucur received a fellowship from the Lady TATA Memorial Trust, London, UK. Maria Sinziana Muraru received a fellowship from Romanian Ministry of Education and Research. This work was also supported by the National Institutes of Health Grants CA105306, CA131664, and HL080192 (to Roya Khosravi-Far).

Conflict of interest

The authors declare no conflict of interest or competing financial interests.

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Authors and Affiliations

  1. Department of Pathology, Harvard Medical School and Beth Israel Deaconess Medical Center, 330 Brookline Ave, Boston, MA, 02215, USA

    Octavian Bucur, Bodvael Pennarun, Andreea Lucia Stancu, Monica Nadler, Maria Sinziana Muraru, Thierry Bertomeu & Roya Khosravi-Far

  2. Department of Molecular Cell Biology, Institute of Biochemistry of the Romanian Academy, Bucharest, Romania

    Octavian Bucur

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  1. Octavian Bucur
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  2. Bodvael Pennarun
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  3. Andreea Lucia Stancu
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  4. Monica Nadler
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  5. Maria Sinziana Muraru
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  6. Thierry Bertomeu
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  7. Roya Khosravi-Far
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Correspondence to Roya Khosravi-Far.

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Octavian Bucur, Bodvael Pennarun, and Andreea Lucia Stancu contributed equally to this work.

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Bucur, O., Pennarun, B., Stancu, A.L. et al. Poor antibody validation is a challenge in biomedical research: a case study for detection of c-FLIP. Apoptosis 18, 1154–1162 (2013). https://doi.org/10.1007/s10495-013-0880-0

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