Hay, J. , Moles, M. W., Cassels, J. and Michie, A. M. (2019) Subcellular fractionation of primary chronic lymphocytic leukemia cells to monitor nuclear/ cytoplasmic protein trafficking. Journal of Visualized Experiments(152), e60426. (doi: 10.3791/60426) (PMID:31710039)
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Abstract
Nuclear export of macromolecules is often deregulated in cancer cells. Tumor suppressor proteins, such as p53, can be rendered inactive due to aberrant cellular localization disrupting their mechanism of action. The survival of chronic lymphocytic leukaemia (CLL) cells, among other cancer cells, is assisted by the deregulation of nuclear to cytoplasmic shuttling, at least in part through deregulation of the transport receptor XPO1 and the constitutive activation of PI3K-mediated signaling pathways. It is essential to understand the role of individual proteins in the context of their intracellular location to gain a deeper understanding of the role of such proteins in the pathobiology of the disease. Furthermore, identifying processes that underlie cell stimulation and the mechanism of action of specific pharmacological inhibitors, in the context of subcellular protein trafficking, will provide a more comprehensive understanding of the mechanism of action. The protocol described here enables the optimization and subsequent efficient generation of nuclear and cytoplasmic fractions from primary chronic lymphocytic leukemia cells. These fractions can be used to determine changes in protein trafficking between the nuclear and cytoplasmic fractions upon cell stimulation and drug treatment. The data can be quantified and presented in parallel with immunofluorescent images, thus providing robust and quantifiable data.
Item Type: | Articles |
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Status: | Published |
Refereed: | Yes |
Glasgow Author(s) Enlighten ID: | Michie, Professor Alison and Moles, Mr Michael and Hay, Dr Jodie and Cassels, Miss Jennifer |
Authors: | Hay, J., Moles, M. W., Cassels, J., and Michie, A. M. |
College/School: | College of Medical Veterinary and Life Sciences > School of Cancer Sciences College of Medical Veterinary and Life Sciences > School of Infection & Immunity |
Journal Name: | Journal of Visualized Experiments |
Publisher: | JoVE |
ISSN: | 1940-087X |
ISSN (Online): | 1940-087X |
Copyright Holders: | Copyright © 2019 Journal of Visualized Experiments |
First Published: | Journal of Visualized Experiments (152):e60426 |
Publisher Policy: | Reproduced under a Creative Commons license |
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