ESTROGEN RECEPTORS. METHODS AND PROTOCOLS

1. The Estrogen Receptors: An Overview from Different Perspectives
2. Competitive Binding Assay for the G-Protein-Coupled Receptor 30 (GPR30) or G-Protein-Coupled Estrogen Receptor (GPER)
3. The Use of Real-Time Reverse Transcription-PCR for Assessing Estrogen Receptor and Estrogen-Responsive Gene Expression
4. Bioinformatics Analysis of Estrogen-Responsive Genes
5. Electrophoretic Mobility Shift Assay (EMSA) and Supershift Assay of Cytochrome P450 2B6 in Response to Estrogen
6. Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors
7. Chromatin Immunoprecipitation with Estrogen Receptor 1 and the Promoter of
8. Chromatin Immunoprecipitation-Sequencing (ChIP-seq) for Mapping of Estrogen Receptor-Chromatin Interactions in Breast Cancer
9. RNA-Seq Experiment and Data Analysis
10. DNA Microarray Analysis of Estrogen-Responsive Genes
11. Shotgun Proteomics Analysis of Estrogen Effects in the Uterus Using Two-Dimensional Liquid Chromatography and Tandem Mass Spectrometry
12. Assessment of Protein Expression by Proximity Ligation Assay in the Nonhuman Primate Endometrium, Placenta, and Fetal Adrenal in Response to Estrogen
13. Colocalization of Estrogen Receptors with the Fluorescent Tamoxifen Derivative, FLTX1, Analyzed by Confocal Microscopy
14. Live-Cell Imaging of the Estrogen Receptor by Total Internal Reflection Fluorescence Microscopy
15. In Situ Hybridization of Estrogen Receptors a and ß and GPER in the Human Testis
16. Purification of Histone Lysine Methyltransferase SMYD2 and Co-Crystallization with a Target Peptide from Estrogen Receptor a
17. Gold Nanoparticle-Based Förster Resonance Energy Transfer (FRET) Analysis of Estrogen Receptor: DNA Interaction
18. Analysis of Interaction of Estradiol with Estrogen Receptor by NMR Spectroscopy
19. Application of Circular Dichroism Spectroscopy to the Analysis of the Interaction Between the Estrogen Receptor Alpha and Coactivators: The Case of Calmodulin
20. Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor a and Specificity Protein 1 with Composite DNA Elements
21. Tips and Tricks for Successful Application of Statistical Methods to Biological Data
22. The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites
23. Selective Estrogen Receptor Modulators and the Tissue-Selective Estrogen Complex: Analysis of Cell Type-Specific Effects Using In Vivo Imaging of a Reporter Mouse Model
24. Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model
25. Comparison of the Effects of the Selective Estrogen Receptor Modulators Ospemifene, Raloxifene, and Tamoxifen on Breast Tissue in Ex Vivo Culture
26. Estrogen Receptor Agonists and Antagonists in the Yeast Estrogen Bioassay
27. Silencing Estrogen Receptor-a with siRNA in the Intact Rodent Brain
28. Silencing Estrogen Receptor-ß with siRNA in Cultured Cells
29. Experimental Procedures for Demonstration of MicroRNA Mediated Enhancement of Functional Neuroprotective Effects of Estrogen Receptor Agonists
30. Expression Profiles of Estrogen-Regulated MicroRNAs in Breast Cancer Cells
31. Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR
32. Detection and Functional Analysis of Estrogen Receptor a Phosphorylated at Serine 216 in Mouse Neutrophils
33. Estrogen Receptor-a Knockout Mice
34. Detection of Endogenous Selective Estrogen Receptor Modulators such as 27-Hydroxycholesterol
35. Phytoestrogens Activate the Estrogen Receptor in HepG2 Cells
36. Detection of the Phosphorylation of the Estrogen Receptor a as an Outcome of GPR30 Activation
37. GPER Mediates Non-Genomic Effects of Estrogen
38. GPER/GPR30 Knockout Mice: Effects of GPER on Metabolism
39. Analysis of G-Protein Coupled Receptor 30 (GPR30) on Endothelial Inflammation
40. Atherosclerosis and Vascular Biologic Responses to Estrogens: Histologic, Immunohistochemical, Biochemical, and Molecular Methods
41. Assessing Direct Vascular Actions of Estrogens
42. Molecular Design, Synthesis, and Evaluation of SNIPER(ER) That Induces Proteasomal Degradation of ERa
43. Regulation of Activation Induced Deaminase (AID) by Estrogen

This volume examines a wide array of vital technologies for advancing our understanding of the receptor-mediated actions of estrogen. The protocols in this book range from standard methods and vital laboratory workhorses, such as receptor binding assays and western blot, to newer technologies such as RNAseq and proximity ligation assay. Chapters also discuss protocols from a broad range of tissue types to demonstrate the variety of estrogen receptor effects. 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.
Cutting-edge and thorough, Estrogen Receptors: Methods and Protocols is a detailed and helpful resource for scientists who are intrigued by the many facets of estrogen.

Features
• Includes cutting-edge methods and protocols
• Provides step-by-step detail essential for reproducible results
• Contains key notes and implementation advice from the experts

Author
Kathleen (Kathy) Eyster Professor of Physiology and Pharmacology Sanford School of Medicine of the University of South Dakota 605-677-5159