, Volume 18, Issue 10, pp 1154–1162

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

  • Octavian Bucur
  • Bodvael Pennarun
  • Andreea Lucia Stancu
  • Monica Nadler
  • Maria Sinziana Muraru
  • Thierry Bertomeu
  • Roya Khosravi-Far
Original Paper


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.


c-FLIP Apoptosis Antibodies Methods Technologies 



TNF-related apoptosis inducing ligand


Death-inducing signaling complex


Tumor necrosis factor


Cellular FLICE-inhibitory protein


Mitogen-activated protein kinases


Extracellular signal-regulated kinase


  1. 1.
    Francia G, Kerbel RS (2010) Raising the bar for cancer therapy models. Nat Biotechnol 28(6):561–562PubMedCrossRefGoogle Scholar
  2. 2.
    Begley CG, Ellis LM (2012) Drug development: raise standards for preclinical cancer research. Nature 483(7391):531–533PubMedCrossRefGoogle Scholar
  3. 3.
    Petricoin EF, Zoon KC, Kohn EC, Barrett JC, Liotta LA (2002) Clinical proteomics: translating benchside promise into bedside reality. Nat Rev Drug Discov 1(9):683–695PubMedCrossRefGoogle Scholar
  4. 4.
    Plati J, Bucur O, Khosravi-Far R (2011) Apoptotic cell signaling in cancer progression and therapy. Integr Biol (Camb) 3(4):279–296CrossRefGoogle Scholar
  5. 5.
    Golks A, Brenner D, Fritsch C, Krammer PH, Lavrik IN (2005) c-FLIPR, a new regulator of death receptor-induced apoptosis. J Biol Chem 280(15):14507–14513PubMedCrossRefGoogle Scholar
  6. 6.
    Oztürk S, Schleich K, Lavrik IN (2012) Cellular FLICE-like inhibitory proteins (c-FLIPs): fine-tuners of life and death decisions. Exp Cell Res 318(11):1324–1331PubMedCrossRefGoogle Scholar
  7. 7.
    Ewald F, Ueffing N, Brockmann L, Hader C, Telieps T, Schuster M, Schulz WA, Schmitz I (2011) The role of c-FLIP splice variants in urothelial tumours. Cell Death Dis 2:e245PubMedCrossRefGoogle Scholar
  8. 8.
    den Hollander MW, Gietema JA, de Jong S, Walenkamp AM, Reyners AK, Oldenhuis CN et al (2013) Translating TRAIL-receptor targeting agents to the clinic. Cancer Lett 332(2):194–201CrossRefGoogle Scholar
  9. 9.
    Bucur O, Ray S, Bucur MC, Almasan A (2006) APO2 ligand/tumor necrosis factor-related apoptosis-inducing ligand in prostate cancer therapy. Front Biosci 11:1549–1568PubMedCrossRefGoogle Scholar
  10. 10.
    Zhang X, Jin TG, Yang H, DeWolf WC, Khosravi-Far R, Olumi AF (2004) Persistent c-FLIP(L) expression is necessary and sufficient to maintain resistance to tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in prostate cancer. Cancer Res 64(19):7086–7091PubMedCrossRefGoogle Scholar
  11. 11.
    Korkolopoulou P, Saetta AA, Levidou G, Gigelou F, Lazaris A, Thymara I et al (2007) c-FLIP expression in colorectal carcinomas: association with Fas/FasL expression and prognostic implications. Histopathology 51(2):150–156PubMedCrossRefGoogle Scholar
  12. 12.
    Chen LC, Chung IC, Hsueh C, Tsang NM, Chi LM, Liang Y, Chen CC, Wang LJ, Chang YS (2010) The antiapoptotic protein, FLIP, is regulated by heterogeneous nuclear ribonucleoprotein K and correlates with poor overall survival of nasopharyngeal carcinoma patients. Cell Death Differ 17(9):1463–1473PubMedCrossRefGoogle Scholar
  13. 13.
    Du X, Bao G, He X, Zhao H, Yu F, Qiao Q, Lu J, Ma Q (2009) Expression and biological significance of c-FLIP in human hepatocellular carcinomas. J Exp Clin Cancer Res 20(28):24CrossRefGoogle Scholar
  14. 14.
    Plati J, Bucur O, Khosravi-Far R (2008) Dysregulation of apoptotic signaling in cancer: molecular mechanisms and therapeutic opportunities. J Cell Biochem 104(4):1124–1149PubMedCrossRefGoogle Scholar
  15. 15.
    Longley DB, Wilson TR, McEwan M, Allen WL, McDermott U, Galligan L et al (2006) c-FLIP inhibits chemotherapy-induced colorectal cancer cell death. Oncogene 25(6):838–848PubMedCrossRefGoogle Scholar
  16. 16.
    Quintavalle C, Incoronato M, Puca L, Acunzo M, Zanca C, Romano G et al (2010) c-FLIPL enhances anti-apoptotic Akt functions by modulation of Gsk3β activity. Cell Death Differ 17(12):1908–1916PubMedCrossRefGoogle Scholar
  17. 17.
    Bivona TG, Hieronymus H, Parker J, Chang K, Taron M, Rosell R et al (2011) FAS and NF-κB signalling modulate dependence of lung cancers on mutant EGFR. Nature 471(7339):523–526PubMedCrossRefGoogle Scholar
  18. 18.
    Oberst A, Dillon CP, Weinlich R, McCormick LL, Fitzgerald P, Pop C et al (2011) Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature 471(7338):363–367PubMedCrossRefGoogle Scholar
  19. 19.
    Peter ME (2011) Programmed cell death: apoptosis meets necrosis. Nature 471(7338):310–312PubMedCrossRefGoogle Scholar
  20. 20.
    Schmidt M, Hupe M, Endres N, Raghavan B, Kavuri S, Geserick P et al (2010) The contact allergen nickel sensitizes primary human endothelial cells and keratinocytes to TRAIL-mediated apoptosis. J Cell Mol Med 14(6B):1760–1776PubMedCrossRefGoogle Scholar
  21. 21.
    Ding J, Polier G, Köhler R, Giaisi M, Krammer PH, Li-Weber M (2012) Wogonin and related natural flavones overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein resistance of tumors by down-regulation of c-FLIP protein and up-regulation of TRAIL receptor 2 expression. J Biol Chem 287(1):641–649PubMedCrossRefGoogle Scholar
  22. 22.
    Kerr E, Holohan C, McLaughlin KM, Majkut J, Dolan S, Redmond K et al (2012) Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis. Cell Death Differ 19(8):1317–1327PubMedCrossRefGoogle Scholar
  23. 23.
    Wang X, Viswanath R, Zhao J, Tang S, Hewlett I (2010) Changes in the level of apoptosis-related proteins in Jurkat cells infected with HIV-1 versus HIV-2. Mol Cell Biochem 337(1–2):175–183PubMedGoogle Scholar
  24. 24.
    Frese S, Brunner T, Gugger M, Uduehi A, Schmid RA (2002) Enhancement of Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)-induced apoptosis in non-small cell lung cancer cell lines by chemotherapeutic agents without correlation to the expression level of cellular protease caspase-8 inhibitory protein. J Thorac Cardiovasc Surg 123(1):168–174PubMedCrossRefGoogle Scholar
  25. 25.
    Haimerl F, Erhardt A, Sass G, Tiegs G (2009) Down-regulation of the de-ubiquitinating enzyme ubiquitin-specific protease 2 contributes to tumor necrosis factor-alpha-induced hepatocyte survival. J Biol Chem 284(1):495–504PubMedCrossRefGoogle Scholar
  26. 26.
    Li Z, Jiang J, Wang Z, Zhang J, Xiao M, Wang C et al (2008) Endogenous interleukin-4 promotes tumor development by increasing tumor cell resistance to apoptosis. Cancer Res 68(21):8687–8694PubMedCrossRefGoogle Scholar
  27. 27.
    Stagni V, di Bari MG, Cursi S, Condò I, Cencioni MT, Testi R et al (2008) ATM kinase activity modulates Fas sensitivity through the regulation of FLIP in lymphoid cells. Blood 111(2):829–837PubMedCrossRefGoogle Scholar
  28. 28.
    Ferrarini M, Delfanti F, Gianolini M, Rizzi C, Alfano M, Lazzarin A et al (2008) NF-kappa B modulates sensitivity to apoptosis, proinflammatory and migratory potential in short-versus long-term cultured human gamma delta lymphocytes. J Immunol 181(9):5857–5864PubMedGoogle Scholar
  29. 29.
    Chandrasekaran Y, McKee CM, Ye Y, Richburg JH (2008) Influence of TRP53 status on FAS membrane localization, CFLAR (c-FLIP) ubiquitinylation, and sensitivity of GC-2spd (ts) cells to undergo FAS-mediated apoptosis. Biol Reprod 74(3):560–568CrossRefGoogle Scholar
  30. 30.
    Chandrasekaran Y, Richburg JH (2005) The p53 protein influences the sensitivity of testicular germ cells to mono-(2-ethylhexyl) phthalate-induced apoptosis by increasing the membrane levels of Fas and DR5 and decreasing the intracellular amount of c-FLIP. Biol Reprod 72(1):206–213PubMedCrossRefGoogle Scholar
  31. 31.
    Mitsiades N, Mitsiades CS, Richardson PG, McMullan C, Poulaki V, Fanourakis G et al (2003) Molecular sequelae of histone deacetylase inhibition in human malignant B cells. Blood 101(10):4055–4062PubMedCrossRefGoogle Scholar
  32. 32.
    Vesely DL, Hoffman B, Liebermann DA (2007) Phosphatidylinositol 3-kinase/Akt signaling mediates interleukin-6 protection against p53-induced apoptosis in M1 myeloid leukemic cells. Oncogene 26(21):3041–3050PubMedCrossRefGoogle Scholar
  33. 33.
    Schuster C, Malinowsky K, Liebmann S, Berg D, Wolff C, Tran K et al (2012) Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues. Histopathology 60(6B):E37–E50PubMedCrossRefGoogle Scholar
  34. 34.
    Razumilava N, Bronk SF, Smoot RL, Fingas CD, Werneburg NW, Roberts LR et al (2012) miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma. Hepatology 55(2):465–475PubMedCrossRefGoogle Scholar
  35. 35.
    Mott JL, Bronk SF, Mesa RA, Kaufmann SH, Gores GJ (2008) BH3-only protein mimetic obatoclax sensitizes cholangiocarcinoma cells to Apo2L/TRAIL-induced apoptosis. Mol Cancer Ther 7(8):2339–2347PubMedCrossRefGoogle Scholar
  36. 36.
    Vesely ED, Heilig CW, Brosius FC 3rd (2009) GLUT1-induced cFLIP expression promotes proliferation and prevents apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 297(3):C759–C765PubMedCrossRefGoogle Scholar
  37. 37.
    Guicciardi ME, Bronk SF, Werneburg NW, Gores GJ (2007) cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis. Am J Physiol Gastrointest Liver Physiol 292(5):G1337–G1346PubMedCrossRefGoogle Scholar
  38. 38.
    Guerreiro-Cacais AO, Levitskaya J, Levitsky V (2010) B cell receptor triggering sensitizes human B cells to TRAIL-induced apoptosis. J Leukoc Biol 88(5):937–945PubMedCrossRefGoogle Scholar
  39. 39.
    Järvinen K, Hotti A, Santos L, Nummela P, Hölttä E (2011) Caspase-8, c-FLIP, and caspase-9 in c-Myc-induced apoptosis of fibroblasts. Exp Cell Res 317(18):2602–2615PubMedCrossRefGoogle Scholar
  40. 40.
    Jeudy S, Wardrop KE, Alessi A, Dominov JA (2011) Bcl-2 inhibits the innate immune response during early pathogenesis of murine congenital muscular dystrophy. PLoS One 6(8):e22369PubMedCrossRefGoogle Scholar
  41. 41.
    Jang JH, Moritz W, Graf R, Clavien PA (2008) Preconditioning with death ligands FasL and TNF-alpha protects the cirrhotic mouse liver against ischaemic injury. Gut 57(4):492–499PubMedCrossRefGoogle Scholar
  42. 42.
    Na IK, Lu SX, Yim NL, Goldberg GL, Tsai J, Rao U et al (2010) The cytolytic molecules Fas ligand and TRAIL are required for murine thymic graft-versus-host disease. J Clin Invest 120(1):343–356PubMedCrossRefGoogle Scholar
  43. 43.
    Jani TS, DeVecchio J, Mazumdar T, Agyeman A, Houghton JA (2010) Inhibition of NF-κB signaling by quinacrine is cytotoxic to human colon carcinoma cell lines and is synergistic in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or oxaliplatin. J Biol Chem 285(25):19162–19172PubMedCrossRefGoogle Scholar
  44. 44.
    Singh A, Ye M, Bucur O, Zhu S, Tanya Santos M, Rabinovitz I et al (2010) Protein phosphatase 2A reactivates FOXO3a through a dynamic interplay with 14-3-3 and AKT. Mol Biol Cell 21(6):1140–1152PubMedCrossRefGoogle Scholar
  45. 45.
    Ray S, Bucur O, Almasan A (2005) Sensitization of prostate carcinoma cells to Apo2L/TRAIL by a Bcl-2 family protein inhibitor. Apoptosis 10(6):1411–1418PubMedCrossRefGoogle Scholar
  46. 46.
    Mori T, Doi R, Kida A, Nagai K, Kami K, Ito D et al (2007) Effect of the XIAP inhibitor Embelin on TRAIL-induced apoptosis of pancreatic cancer cells. J Surg Res 142(2):281–286PubMedCrossRefGoogle Scholar
  47. 47.
    Kola I, Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 3(8):711–715PubMedCrossRefGoogle Scholar
  48. 48.
    Prinz F, Schlange T, Asadullah K (2011) Believe it or not: how much can we rely on published data on potential drug targets? Nat Rev Drug Discov 10(9):712PubMedCrossRefGoogle Scholar
  49. 49.
    Mullard A (2011) Reliability of ‘new drug target’ claims called into question. Nat Rev Drug Discov 10(9):643–644PubMedCrossRefGoogle Scholar
  50. 50.
    Lavrik IN, Krammer PH (2012) Regulation of CD95/Fas signaling at the DISC. Cell Death Differ 19(1):36–41PubMedCrossRefGoogle Scholar
  51. 51.
    Jensen BC, Swigart PM, Simpson PC (2009) Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific. Naunyn Schmiedebergs Arch Pharmacol 379(4):409–412PubMedCrossRefGoogle Scholar
  52. 52.
    Herber DL, Severance EG, Cuevas J, Morgan D, Gordon MN (2004) Biochemical and histochemical evidence of nonspecific binding of alpha7nAChR antibodies to mouse brain tissue. J Histochem Cytochem 52(10):1367–1376PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Octavian Bucur
    • 1
    • 2
  • Bodvael Pennarun
    • 1
  • Andreea Lucia Stancu
    • 1
  • Monica Nadler
    • 1
  • Maria Sinziana Muraru
    • 1
  • Thierry Bertomeu
    • 1
  • Roya Khosravi-Far
    • 1
  1. 1.Department of PathologyHarvard Medical School and Beth Israel Deaconess Medical CenterBostonUSA
  2. 2.Department of Molecular Cell BiologyInstitute of Biochemistry of the Romanian AcademyBucharestRomania

Personalised recommendations