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Analyzing Ras-Associated Cell Proliferation Signaling

  • Matthew C. Stout
  • Edgar Asiimwe
  • James R. Birkenstamm
  • Su Yeon Kim
  • Paul M. CampbellEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1170)

Abstract

Ras-dependent signaling is an important regulator of cell cycle progression, proliferation, senescence, and apoptosis. Several of the downstream effectors of Ras play dual roles in each of these processes. Under one set of conditions, they promote cell cycle progression and proliferation; yet, in a different paradigm, they drive cell cycle arrest and apoptosis. Furthermore, there is cross talk between certain downstream effectors of Ras including the PI3K–AKT and Raf–MEK–ERK pathways. Here we describe a series of experiments used to dissect the effect of different Ras-dependent signaling pathways on cell cycle progression, proliferation, senescence, and apoptosis. Furthermore, we highlight the importance of consistent growth conditions of cells in culture when studying Ras-dependent signaling as we show that the activation of downstream effectors of Ras changes with the confluency at which the cells are grown.

Key words

Ras Cell cycle Proliferation Senescence Anoikis Pulldown assay Confluency 

Notes

Acknowledgements

We would like to thank Channing J. Der for the kind gift of the GTPase-binding domain pGEX constructs used in the pulldown assays.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Matthew C. Stout
    • 1
  • Edgar Asiimwe
    • 1
  • James R. Birkenstamm
    • 1
  • Su Yeon Kim
    • 1
  • Paul M. Campbell
    • 1
    Email author
  1. 1.Department of Pharmacology and PhysiologyDrexel University College of MedicinePhiladelphiaUSA

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