Main description:

This volume provides state-of-the-art techniques for studying various aspects of cholesterol homeostasis, including its uptake, synthesis and efflux from the cell, as well as its trafficking within the cell. Chapters also cover techniques for studying the regulation of cholesterol homeostasis at both the transcriptional and post-translational levels, as well as studying the membrane topology and structure of cholesterol-related proteins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and practical, Cholesterol Homeostasis: Methods and Protocols aims to provide key techniques in tackling the investigation of cholesterol homeostasis.

Contents:

1. An Overview of Cholesterol Homeostasis

Ingrid C. Gelissen and Andrew J. Brown

2. Hybrid In Silico/In Vitro Approaches for the Identification of Functional Cholesterol Binding Domains in Membrane Proteins

Coralie Di Scala and Jacques Fantini

3. Structural Stringency of Cholesterol for Membrane Protein Function utilizing Stereoisomers as Novel Tools: A Review

Md. Jafurulla and Amitabha Chattopadhyay

4. Manipulating Cholesterol Status within Cells

Winnie Luu, Ingrid C. Gelissen, and Andrew J. Brown

5. Assaying Low-Density-Lipoprotein (LDL) Uptake into Cells Anke Loregger, Jessica Kristine Nelson, and Noam Zelcer

6. The use of L-sIDOL Transgenic Mice as a Murine Model to Study Hypercholesterolaemia and Atherosclerosis

Eser J. Zerenturk and Anna C. Calkin

7. CRISPR/Cas9-mediated Generation of Niemann-Pick C1 Knockout Cell Line

Ximing Du, Ivan Lukmantara, Hongyuan Yang

8. Quantitative Measurement of Cholesterol in Cell Population Using Flow Cytometry and Fluorescent Perfringolysin O*

Jian Li, Peter L. Lee, and Suzanne R. Pfeffe

9. Transport Assays for Sterol-binding Proteins: Stopped-flow Fluorescence Methods for Investigating Intracellular Cholesterol Transport Mechanisms of NPC2 Protein

Leslie A McCauliff1 and Judith Storch

10. Synthesis and Live-cell Imaging of Fluorescent Sterols for Analysis of Intracellular Cholesterol Transport

Maciej Modzel, Frederik W. Lund, and Daniel Wustner

11. Measurement of Cholesterol Transfer from Lysosome to Peroxisomes Using an In Vitro Reconstitution Assay

Jie Luo, Ya-Cheng Liao, Jian Xiao, and Bao-Liang Song

12. Measurement of Mitochondrial Cholesterol Import using a Mitochondria-targeted CYP11A1 Fusion Construct

Barry Edward Kennedy, Mark Charman, and Barbara Karten

13. Identifying Sterol Response Elements within Promoters of Genes

Laura J. Sharpe and Andrew J. Brown

14. Membrane Extraction of HMG CoA Reductase as Determined by Susceptibility of Lumenal Epitope to In Vitro Protease Digestion

Lindsey L. Morris and Russell A. DeBose-Boyd

15. Determining the Topology of Membrane-bound Proteins using PEGylation

Vicky Howeand Andrew J Brown

16. Measuring Activity of Cholesterol Synthesis Enzymes Using Gas Chromatography/Mass Spectrometry

Anika V. Prabhu, Winnie Luu, and Andrew J. Brown

17. Sterol Analysis by Quantitative Mass Spectrometry

Andrew M. Jenner and Simon H.J. Brown

18. Measurement of Rates of Cholesterol and Fatty Acid Synthesis in vivo using Tritiated Water

Adam M. Lopez, Jen-Chieh Chuang and Stephen D. Turley

19. Methods for Monitoring ABCA1-dependent Sterol Release

Yoshio Yamauchi, Shinji Yokoyama, and Ta-Yuan Chang

20. ABC-transporter Mediated Sterol Export from Cells using Radio-Labeled Sterols

Alryel Yang and Ingrid C. Gelissen

21. Measurement of Macrophage-specific In Vivo Reverse Cholesterol Transport in Mice

M. Kockx, W. Jessup and L. Kritharides