Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening

Ivana Gaziova, Robert A. Davey, Lisa Elferink

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

The tyrosine kinase MET, a receptor for hepatocyte growth factor, is a key regulator for normal development and organ renewal via stem cell maintenance. Dysregulated MET signaling contributes to tumor progression and metastasis and is considered a potent therapeutic target for a growing number of malignancies. Toward that goal it is critical to develop high-throughput assays to identify candidate regulators for the termination of MET signaling. We describe here a rapid and efficient method for identifying cellular factors required for MET ubiquitination, which utilizes high-throughput RNA interference screening (HT-siRNA) with a receptor internalization assay and an In-Cell ELISA in a 96-well format. The assay is amenable to a large array of cell surface proteins as well as genome-wide siRNA libraries, with high signal-to- background ratio and low well-to-well variability.

Original languageEnglish (US)
Pages (from-to)381-394
Number of pages14
JournalMethods in molecular biology (Clifton, N.J.)
Volume1270
DOIs
StatePublished - 2015
Externally publishedYes

Fingerprint

Endocytosis
Small Interfering RNA
Proto-Oncogene Proteins c-met
Ubiquitination
RNA Interference
Protein-Tyrosine Kinases
Neoplasms
Membrane Proteins
Enzyme-Linked Immunosorbent Assay
Maintenance
Genome
Neoplasm Metastasis
Therapeutics
Cell Self Renewal

Keywords

  • HeLa cells
  • Hepatocyte growth factor (HGF)
  • MET
  • Receptor endocytosis
  • RNA interference (siRNA)
  • Ube3C
  • Ubiquitination

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Identification of factors regulating MET receptor endocytosis by high-throughput siRNA screening. / Gaziova, Ivana; Davey, Robert A.; Elferink, Lisa.

In: Methods in molecular biology (Clifton, N.J.), Vol. 1270, 2015, p. 381-394.

Research output: Contribution to journalArticle

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