Advertisement

Western Blotting Analysis as a Tool to Study Receptor Tyrosine Kinases

  • Serena GermanoEmail author
  • Lorraine O’Driscoll
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 784)

Abstract

Receptor tyrosine kinases (RTKs) are involved in critical aspects of cell physiology ranging from cell ­survival, proliferation, growth, migration, and differentiation. A tight control of the extent and duration of signals elicited by activated RTKs is crucial for preventing over-stimulation, which can ultimately lead to unrestrained proliferative ability and neoplastic growth. Ligand-induced downregulation of RTKs has emerged as a key negative regulatory mechanism that can accomplish signaling attenuation, by removing activated receptors from the cell surface and committing them to degradation. The ability of RTKs to escape from ligand-induced downregulation has been reported as a recurrent mechanism of oncogenic deregulation in cancer.

Western blotting procedures have been extensively proven as straightforward assays to evaluate ­protein expression levels and have been widely applied to study RTKs downregulation.

Key words

Protein Western blotting Receptor tyrosine kinases 

Notes

Acknowledgments

Preparation of this chapter was supported by the Health Research Board of Ireland (Grant RP/2006/77); Trinity College Dublin’s Start-Up Funds for New Academics 2008/2009 and Science Foundation Ireland’s funding of Molecular Therapeutics for Cancer, Ireland (08/SRC/B1410).

References

  1. 1.
    Blume-Jensen, P., Hunter, T. (2001) Oncogenic kinase signalling. Nature. 411 355365.PubMedCrossRefGoogle Scholar
  2. 2.
    Dikic, I., Giordano, S. (2003) Negative receptor signalling. Curr Opin Cell Biol. 15 128–135.PubMedCrossRefGoogle Scholar
  3. 3.
    Peschard, P., Park, M. (2003) Escape from Cbl-mediated downregulation: a recurrent theme for oncogenic deregulation of receptor tyrosine kinases. Cancer Cell. 3 519–523.PubMedCrossRefGoogle Scholar
  4. 4.
    Gaudino, G., Follenzi, A., Naldini, L., Collesi, C., Santoro, M., Gallo, K.A., Godowski, P.J., Comoglio, P.M. (1994) RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP. EMBO J. 13 3524–3532.PubMedGoogle Scholar
  5. 5.
    Wang, M.H., Ronsin, C., Gesnel, M.C., Coupey, L., Skeel, A., Leonard, E.J., Breathnach, R. (1994) Identification of the ron gene product as the receptor for the human macrophage stimulating protein. Science. 266 117119.PubMedCrossRefGoogle Scholar
  6. 6.
    Germano, S., Gaudino, G. (2008) Molecular targets in cancer therapy: the Ron approach. Oncology Rev. 1 215224.CrossRefGoogle Scholar
  7. 7.
    Wang, M.H., Wang, D., Chen, Y.Q. (2003) Oncogenic and invasive potentials of human macrophage-stimulating protein receptor, the RON receptor tyrosine kinase. Carcinogenesis. 24 1291–1300.PubMedCrossRefGoogle Scholar
  8. 8.
    Peace, B.E., Toney-Earley, K., Collins, M.H., Waltz, S.E. (2005) Ron receptor signaling augments mammary tumor formation and metastasis in a murine model of breast cancer. Cancer Res. 65 1285–1293.PubMedCrossRefGoogle Scholar
  9. 9.
    Willett, C.G., Wang, M.H., Emanuel, R.L., Graham, S.A., Smith, D.I., Shridhar, V., Sugarbaker, D.J., Sunday, M.E. (1998) Macrophage-stimulating protein and its receptor in non-small-cell lung tumors: induction of receptor tyrosine phosphorylation and cell migration. Am J Respir Cell Mol Biol. 18 489–496.PubMedGoogle Scholar
  10. 10.
    Zhou, Y.Q., He, C., Chen, Y.Q., Wang, D., Wang, M.H. (2003) Altered expression of the RON receptor tyrosine kinase in primary human colorectal adenocarcinomas: generation of different splicing RON variants and their oncogenic potential. Oncogene. 22 186–97.PubMedCrossRefGoogle Scholar
  11. 11.
    Cheng, H.L., Liu, H.S., Lin, Y.J., Chen, H.H., Hsu, P.Y., Chang, T.Y., Ho, C.L., Tzai, T.S., Chow, N.H. (2005) Co-expression of RON and MET is a prognostic indicator for patients with transitional-cell carcinoma of the bladder. Br J Cancer. 92 1906–1914.PubMedCrossRefGoogle Scholar
  12. 12.
    Germano, S., Barberis, D., Santoro, M.M., Penengo, L., Citri, A., Yarden, Y., Gaudino, G. (2006) Geldanamycins trigger a novel Ron degradative pathway, hampering oncogenic signaling. J Biol Chem. 281 2171021719.PubMedCrossRefGoogle Scholar
  13. 13.
    Burnette, W.N. (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein. A. Anal Biochem. 112 195203.CrossRefGoogle Scholar
  14. 14.
    Harlow, E. and Lane, D. (1999) Using antibodies: A laboratory manual, Cold Spring Harbor Laboratory Press.Google Scholar
  15. 15.
    Martin, C.S. and Bronstein, I. (1994) Imaging of chemiluminescent signals with cooled CCD camera systems. J Biolumin Chemilumin. 9 145153.PubMedCrossRefGoogle Scholar
  16. 16.
    MacPhee, D.J. (2009) Methodological considerations for improving Western blot analysis. J Pharmacol Toxicol Methods. 61(2) 171177.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.School of Pharmacy and Pharmaceutical SciencesTrinity College DublinDublinIreland

Personalised recommendations