Endosome signalling. Part B / edited by Michael Conn P.

San Diego, California : Academic Press, 2014.
1 online resource (517 p.)
First edition.
Methods in enzymology ; v. 535.
Methods in Enzymology ; Volume 535

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Cell interaction.
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This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This is the second of two volumes on endosome signaling and includes chapters on such topics as measurement of entry into the endosomal compartment by multi-parametric image analysis, assessment of peptide internalization and endosomal signaling, and VEGF-A in endosomal signaling. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers endosome signalingContai
Front Cover; Endosome Signaling Part B; Copyright; Contents; Contributors; Preface; Section I: Signaling; Chapter One: Assessment of Insulin Proteolysis in Rat Liver Endosomes: Its Relationship to Intracellular Insulin Signaling; 1. Introduction; 2. Assays for Insulin Degradation; 2.1. Trichloroacetic acid precipitation assay; 2.2. Reverse-phase HPLC assay using radiolabeled insulin; 2.3. Reverse-phase HPLC assay using native insulin; 3. Assay for Endosomal Proteolysis of Insulin; 3.1. In vivo studies; 3.2. Cell-free endosome studies; 3.3. In vitro studies
4. Sites of Cleavage and Degradation Products of Insulin within Endosomes5. Endosomal Insulinases; 5.1. Cathepsin D; 5.2. Neutral Arg aminopeptidase; 5.3. Insulin-degrading enzyme; 6. Effects of Insulin and Slowly Processed Insulin Analogs on Insulin Signaling in Liver Endosomes; 6.1. Endosomal IR tyrosine phosphorylation; 6.1.1. Procedure; 6.2. Translocation of insulin-signaling proteins to endosomes; 6.2.1. Procedure; 7. Involvement of IR Endocytosis in Positive Regulation of Insulin Signaling; References
Chapter Two: A Bimolecular Fluorescent Complementation Screen Reveals Complex Roles of Endosomes in Ras-Mediated Signaling1. Introduction; 1.1. Ras protein and growth factor signaling; 1.2. RTK and Ras internalization; 1.3. Ras interaction with Cdc42 in the endosome is required for efficient transformation; 1.4. Using BiFC to seek and study new Ras effectors; 1.5. Promoting Ras recycling and transformation by components of the ESCRT-III complex; 2. Detect Ras-effector Interactions by BiFC; 2.1. General considerations for using BiFC to study Ras; 2.2. Detect Ras-effector binding by FACS
2.3. Detect Ras-effector binding by microscopy3. Screen for Ras-binding Proteins by BiFC; 3.1. Create ``baits ́ ́ and negative controls; 3.2. Create cDNA libraries; 3.3. Initial screen for binding partners by FACS; 3.4. Follow-up screens to confirm the binding; 3.5. Functional validation; 4. Concluding Remarks; Acknowledgments; References; Chapter Three: TGFβ in Endosomal Signaling; 1. Introduction; 2. Endocytosis; 2.1. Isolation of membrane rafts; 2.1.1. Detergent-free membrane raft isolation protocol; 3. The Role of the Early Endosome in TGFβ Signal Transduction
3.1. Fluorescence techniques to follow cell-surface receptor endocytosis3.2. Biotinylation of TGFβ; Biotinylation of TGFβ; 4. Readouts of TGFβ Signal Transduction; 4.1. Smad2 phosphorylation; Acknowledgments; References; Chapter Four: Annexins and Endosomal Signaling; 1. Introduction; 2. Isolation of Endocytic Compartments; 2.1. Isolation of endocytic fractions from rat liver; 2.1.1. Characterization of the recycling endocytic compartment (RRC); 2.2. Isolation of endocytic fractions from CHO cells; 2.2.1. Early and late endosomes; 3. Targeting Raf-1 Signaling to Early Endosomes
3.1. Expression of CFP-2xFYVE-Raf-1 and Raf-1 activation analysis
Description based upon print version of record.
Includes bibliographical references and index.
Description based on online resource; title from PDF title page (ebrary, viewed January 22, 2014).
Conn, P. Michael.