Description
SPRINGER Phospholipid Signaling Protocols 1998 Edition by BIRD, IAN (ED.)
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. Part I. Monitoring of Activation of Phospholipid-Derived Cell Signaling PathwaysPhosphoinositidase C Activation Assay I: Cell Labeling, Stimulation, and Recovery of Cellular [3H] Phosphoinositides and [3H] PhosphoinositolsIan M. BirdPhosphoinositidase C Activation Assay II: Simple Analysis of Recovered Cellular Phosphoinositides and PhosphoinositolsIan M. BirdPhosphoinositidase C Activation Assay III: HPLC Analysis of Cellular Phosphoinositides and PhosphoinositolsIan M. BirdMeasurement of Phosphoinositols and Phosphoinositides Using Radio High-Performance Liquid Chromatography Flow DetectionLubo Zhang and Iain L. O. BuxtonPreparation of [3H]Phosphoinositol Standards and Conversion of [3H] Phosphoinositides to [3H] PhosphoinositolsIan M. BirdInositol 1,4,5-Trisphosphate Mass AssayLubo ZhangMeasurement of Cellular Diacylglycerol ContentWendy Bollinger Bollag and Richard D. GrinerPhosphatidylinositol 4-Kinases: Assays and Product AnalysisRachel E. Meyers and Lewis C. CantleyPhosphatidylinositol(3,4,5)Trisphosphate (Ptdins(3,4,5)P3) Mass Measurement Using a Radioligand Displacement AssayJeroen van der Kaay, Pete J. Cullen, and C. Peter DownesDetection of Phosphatidylinositol-4-Phosphate 5-Kinase Activity Using Thin-Layer ChromatographyGregory J. Parker, Joost C. Loijens, and Richard A. AndersonDetermination of Phospholipase C- or Phospholipase D-Catalyzed Phosphatidylcholine HydrolysisMichael J. O. Wakelam and Trevor R. PettittMeasurement of Phospholipase D ActivityWendy Bollinger BollagMonitoring of Phospholipase A2 Activation in Cultured Cells Using Tritiated Arachidonic AcidSandie I. Briand, Sylvie G. Bernier, and Gaetan GuillemetteAssay of Cellular Diacylglycerol and Monoacylglycerol LipasesBryan A.WolfIsotopic Efflux Studies as Indices of Phospholipase ActivationJacob Vadakekalam and Stewart MetzExtraction and Measurements of Prostanoids and Leukotrienes by RadioimmunoassaysBruno Battistini, Serge Picard, Pierre Borgeat, and Pierre SiroisMeasurement of Prostanoids, Leukotrienes, and Isoprostanes By Enzyme ImmunoassaysBruno Battistini, Serge Picard, Pierre Borgeat, and Pierre SiroisMeasurements of Arachidonic Acid Metabolites Derived from the Lipoxygenase Pathways By High-Pressure Liquid ChromatographyPierre Borgeat, Serge Picard, Bruno Battistini, and Pierre SiroisMeasurement of Sphingomyelin and Ceramide Cellular Levels After Sphingomyelinase-Mediated Sphingomyelin HydrolysisPino Santana, Luisa F. Fanjul, and C. M. Ruiz de GalarretaSphingomyelin and Ceramide Mass AssayPino Santana, C. M. Ruiz de Galarreta, and Luisa F. FanjulSphingosine Kinase: Assay and Product AnalysisAna Olivera and Sarah SpiegelAnalytical Methods and Steps to Sample Preparation For Determination of Molecular Species of Fatty AcidsSanda ClejanHPLC Analytical Methods for the Separation of Molecular Species of Fatty Acids In Diacylglycerol and Cellular PhospholipidsSanda ClejanAnalysis of Molecular Species of Cellular Sphingomyelins and CeramidesSanda ClejanPart II. General methods for monitoring changes in levels of key signaling pathway proteins and associated mRNASolubilization and Assay of Cellular and Tissue ProteinCynthia E. ShawWestern Immunoblot AnalysisCynthia E. Shaw and Jing ZhengImmunohistochemistry and ImmunocytochemistryJing ZhengExtraction of Cellular and Tissue RNADaniel S. Millican and Ian M. BirdSize Separation and Quantification of mRNA by Northern AnalysisIan M. BirdPreparation of