Subcellular fractionation of primary chronic lymphocytic leukemia cells to monitor nuclear/ cytoplasmic protein trafficking

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
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Michie, Dr 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 > Institute of Cancer Sciences
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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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
3030910Elucidating the mechanisms that regulate FOXO activity in chronic lymphocytic leukaemia a novel target for therapeutic exploitation?Alison MichieBloodwise (BLOODWIS)18003CS - Paul O'Gorman Leukaemia Research Centre