Phospholipid Signaling Protocols, Softcover reprint of the original 1st ed. 1998 Methods in Molecular Biology Series, Vol. 105

Cell membranes are not, as once believed, inert structures designed to contain the cell contents, but are in fact dynamic structures that are as me- bolically active as the cytosol and other cellular compartments they surround. Thus membranes not only contain mixtures of lipid and phospholipids, but also many proteins both embedded deeply within the membrane structure itself and also more loosely attached on the membrane surfaces. Though many such proteins have long been known to act as transport proteins, ion channels, hormone receptors, G proteins, cytoskeletal anchorage points, and so on, the major advance of recent years is the increasing understanding that the lipids and phospholipids in the membrane bilayer itself are also metabolized to b- logically active products that can diffuse either in the cytosol or in the m- brane bilayer to control the function of other proteins. Thus the concept of lipid-derived second messengers is now firmly established.

Monitoring of Activation of Phospholipid-Derived Cell Signaling Pathways.- Phosphoinositidase C Activation Assay I.- Phosphoinositidase C Activation Assay II.- Phosphoinositidase C Activation Assay III.- Measurement of Phosphoinositols and Phosphoinositides Using Radio High-Performance Liquid Chromatography Flow Detection.- Preparation of [3H]Phosphoinositol Standards and Conversion of [3H]Phosphoinositides to [3H]Phosphoinositols.- Inositol 1,4,5-Trisphosphate Mass Assay.- Measurement of Cellular Diacylglycerol Content.- Phosphatidylinositol 4-Kinases.- Phosphatidylinositol(3,4,5)Trisphosphate (Ptdins(3,4,5)P3) Mass Measurement Using a Radioligand Displacement Assay.- Detection of Phosphatidylinositol-4-Phosphate 5-Kinase Activity Using Thin-Layer Chromatography.- Determination of Phospholipase C-or Phospholipase D-Catalyzed Phosphatidylcholine Hydrolysis.- Measurement of Phospholipase D Activity.- Monitoring of Phospholipase A2 Activation in Cultured Cells Using Tritiated Arachidonic Acid.- Assay of Cellular Diacylglycerol and Monoacylglycerol Lipases.- Isotopic Efflux Studies as Indices of Phospholipase Activation.- Extraction and Measurements of Prostanoids and Leukotrienes by Radioimmunoassays.- Measurements of Prostanoids, Leukotrienes, and Isoprostanes by Enzyme Immunoassays.- Measurements of Arachidonic Acid Metabolites Derived from the Lipoxygenase Pathways by High-Pressure Liquid Chromatography.- Measurement of Sphingomyelin and Ceramide Cellular Levels After Sphingomyelinase-Mediated Sphingomyelin Hydrolysis.- Sphingomyelin and Ceramide Mass Assay.- Sphingosine Kinase.- Analytical Methods and Steps to Sample Preparation for Determination of Molecular Species of Fatty Acids.- HPLC Analytical Methods for the Separation of Molecular Species of Fatty Acids in Diacylglycerol and Cellular Phospholipids.- Analysis of Molecular Species of Cellular Sphingomyelins and Ceramides.- General Methods for Monitoring Changes in Levels of Key Signaling Pathway Proteins and Associated mRNA.- Solubilization and Assay of Cellular and Tissue Protein.- Western Immunoblot Analysis.- Immunohistochemistry and Immunocytochemistry.- Extraction of Cellular and Tissue RNA.- Size Separation and Quantification of mRNA by Northern Analysis.- Preparation of Single-Stranded Antisense cDNA Probes by Asymmetric PCR.- Optimization of a Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Mass Assay for Low-Abundance mRNA.