Related Experiment Video

Updated: Jun 26, 2026

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
10:52

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Published on: January 6, 2016

Assaying endogenous phosphatidylinositol-4-phosphate 5-kinase (PIP5K) activities

Jonathan R Halstead1, Mireille H Snel, Sarah Meeuws

  • 1Division of Cellular Biochemistry, The Netherlands Cancer Institute, Amsterdam.

Methods in Molecular Biology (Clifton, N.J.)
|January 24, 2009
PubMed

Insights

Researchers detail methods to reduce phosphatidylinositol-4-phosphate 5-kinase beta (PIP5Kbeta) expression and measure its activity. This work aids in understanding phosphoinositide signaling in cancer pathways.

Area of Science:

  • Lipid signaling
  • Molecular biology
  • Cancer research

Background:

  • Phosphoinositides are crucial lipid second messengers regulating cancer-related pathways.
  • PtdIns(4,5)P2 is central to phosphoinositide signaling, with its levels controlled by synthesis and degradation enzymes.
  • Phosphatidylinositol-4-phosphate 5-kinases (PIP5K) are key enzymes in PtdIns(4,5)P2 generation.

Purpose of the Study:

  • To describe methods for suppressing human PIP5Kbeta expression using RNA interference (RNAi).
  • To outline techniques for measuring endogenous PIP5Kbeta activity and levels.
  • To facilitate research into the role of PIP5Kbeta in cancer.

Main Methods:

  • RNA interference (RNAi) for suppressing PIP5Kbeta gene expression.
  • Immunoprecipitation to isolate endogenous PIP5Kbeta.
  • Enzyme activity assays to quantify PIP5Kbeta function.
  • Western blotting to determine PIP5Kbeta protein levels.

Main Results:

  • Demonstrated RNAi efficacy in reducing PIP5Kbeta expression.
  • Established a reliable method for immunoprecipitating and assaying endogenous PIP5Kbeta activity.
  • Quantified endogenous PIP5Kbeta levels using Western blotting.

Conclusions:

  • The described methods enable robust investigation of PIP5Kbeta function.
  • These techniques are valuable for studying phosphoinositide dysregulation in cancer.
  • Further research can utilize these methods to explore PIP5Kbeta's role in cancer progression.

More Related Videos

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions10:58

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Published on: July 27, 2017

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes07:26

Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

Published on: October 15, 2016

Related Concept Videos

Phosphoinositides and PIPs01:42

Phosphoinositides and PIPs

Phosphoinositides are a group of phospholipids containing a glycerol backbone with two fatty acid chains and a phosphate attached to a myoinositol sugar ring. The inositol head group extends into the cytoplasm, where it is modified by adding phosphate groups to form phosphatidylinositol phosphates or PIPs.
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...
IP3/DAG Signaling Pathway01:11

IP3/DAG Signaling Pathway

Membrane lipids such as phosphatidylinositol (PI) are precursors for several membrane-bound and soluble second messengers. Specific kinases phosphorylate PI and produce phosphorylated inositol phospholipids. One such inositol phospholipids are the  phosphatidylinositol-4,5 bisphosphate [PI(4,5)P2], present in the inner half of the lipid bilayer. Upon ligand binding, GPCR stimulates Gq proteins to turn on phospholipase Cꞵ. Activated phospholipase Cꞵ cleaves PI(4,5)P2 and produces two-second...
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
The JAK-STAT Signaling Pathway01:20

The JAK-STAT Signaling Pathway

Several cytokine receptors have tightly bound Janus kinase or JAK proteins attached at their cytosolic tail. Small signaling molecules such as cytokines, growth hormones, or prolactins bind to the cytokine receptors and initiate their dimerization. The dimerization brings the cytosolic JAKs together that trans-phosphorylate and activates each other. The activated JAKs now phosphorylate cytosolic tails of the cytokine receptors, which serve as binding sites for adaptor proteins such as  SH2...