Assaying endogenous phosphatidylinositol-4-phosphate 5-kinase (PIP5K) activities
Assaying endogenous phosphatidylinositol-4-phosphate 5-kinase (PIP5K) activities
Phosphoinositides are a family of lipid second messengers interlinked by an extensive and highly regulated network of kinases and phosphatases. The modulation of phosphoinositide profiles can regulate numerous cancer-related pathways, including cell survival, cell proliferation, migration, integrin activation, and transcription. PtdIns(4,5)P2 is at the heart of phosphoinositide signaling; its levels are controlled by enzymes that synthesize it and those that degrade it. Phosphatidylinositol-4-phosphate 5-kinases (PIP5 K) phosphorylate PtdIns4P on the 5-position and constitute the major pathway for the generation of PtdIns(4,5)P2. We will discuss how to suppress the expression of human PIP5 Kbeta using RNAi and how to measure the activity and levels of the endogenous enzyme. We describe a method to immunoprecipitate the endogenous PIP5 Kbeta and to assay its activity. Western blotting with another panel of antibodies is then used to determine the levels of endogenous PIP5 Kbeta in the immunoprecipitates.
Amino Acid Sequence Animals Blotting, Western Cell Line Gene Knockdown Techniques Humans Immunoprecipitation Isoenzymes/analysis/chemistry/genetics/metabolism Molecular Sequence Data Phosphotransferases (Alcohol Group Acceptor)/*analysis/chemistry/genetics/*metabolism RNA Interference
391-402
Halstead, J. R.
019c9dab-c338-4f97-9318-353858ea82ac
Snel, M. H.
68a0a7a3-d3c0-4261-a5df-af0d8499332c
Meeuws, S.
3e8a2a45-93b3-48ef-84b2-d0ca93b25e35
Jones, D. R.
fcbc07f0-6ce7-4ea0-b8b3-f0e6a9581279
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Halstead, J. R.
019c9dab-c338-4f97-9318-353858ea82ac
Snel, M. H.
68a0a7a3-d3c0-4261-a5df-af0d8499332c
Meeuws, S.
3e8a2a45-93b3-48ef-84b2-d0ca93b25e35
Jones, D. R.
fcbc07f0-6ce7-4ea0-b8b3-f0e6a9581279
Divecha, N.
5c2ad0f8-4ce7-405f-8a15-2fc4ab96d787
Halstead, J. R., Snel, M. H., Meeuws, S., Jones, D. R. and Divecha, N.
(2008)
Assaying endogenous phosphatidylinositol-4-phosphate 5-kinase (PIP5K) activities.
Methods in Molecular Biology, 462, .
(doi:10.1007/978-1-60327-115-8_25).
Abstract
Phosphoinositides are a family of lipid second messengers interlinked by an extensive and highly regulated network of kinases and phosphatases. The modulation of phosphoinositide profiles can regulate numerous cancer-related pathways, including cell survival, cell proliferation, migration, integrin activation, and transcription. PtdIns(4,5)P2 is at the heart of phosphoinositide signaling; its levels are controlled by enzymes that synthesize it and those that degrade it. Phosphatidylinositol-4-phosphate 5-kinases (PIP5 K) phosphorylate PtdIns4P on the 5-position and constitute the major pathway for the generation of PtdIns(4,5)P2. We will discuss how to suppress the expression of human PIP5 Kbeta using RNAi and how to measure the activity and levels of the endogenous enzyme. We describe a method to immunoprecipitate the endogenous PIP5 Kbeta and to assay its activity. Western blotting with another panel of antibodies is then used to determine the levels of endogenous PIP5 Kbeta in the immunoprecipitates.
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e-pub ahead of print date: 1 January 2008
Additional Information:
Halstead, Jonathan R Snel, Mireille H Meeuws, Sarah Jones, David R Divecha, Nullin eng Research Support, Non-U.S. Gov't 2009/01/24 Methods Mol Biol. 2009;462:391-402. doi: 10.1007/978-1-60327-115-8_25.
Keywords:
Amino Acid Sequence Animals Blotting, Western Cell Line Gene Knockdown Techniques Humans Immunoprecipitation Isoenzymes/analysis/chemistry/genetics/metabolism Molecular Sequence Data Phosphotransferases (Alcohol Group Acceptor)/*analysis/chemistry/genetics/*metabolism RNA Interference
Identifiers
Local EPrints ID: 480209
URI: http://eprints.soton.ac.uk/id/eprint/480209
ISSN: 1064-3745
PURE UUID: 81005560-2ce1-484e-aa87-e91e4d214b2e
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Date deposited: 01 Aug 2023 17:04
Last modified: 17 Mar 2024 02:59
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Author:
J. R. Halstead
Author:
M. H. Snel
Author:
S. Meeuws
Author:
D. R. Jones
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