Springer protocols feed by Biochemistry 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Detection and Functional Analysis of Estrogen Receptor α Phosphorylated at Serine 216 in Mouse Neutrophils
Serine 216 constitutes a protein kinase C phosphorylation motif located within the DNA binding domain of estrogen receptor α (ERα). In this chapter we present experimental procedures confirming that mouse ERα is phosphorylated at serine 216 in peripheral blood neutrophils and in neutrophils that infiltrate the uterus, as well as the role of phosphoserine 216 in neutrophil migration. A phospho-peptide antibody (αP-S216) was utilized in Western blot, immunohistochemistry, and double immunofluorescence staining to detect this phosphorylation of an endogenous ERα. Both immunohistochemistry (with &...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Estradiol-Induced Transcriptional Regulation of Long Non-Coding RNA, HOTAIR
HOTAIR (HOX antisense intergenic RNA) is a 2.2 kb long non-coding RNA (lncRNA), transcribed from the antisense strand of homeobox C (HOXC) gene locus in chromosome 12. HOTAIR acts as a scaffolding lncRNA. It interacts and guides various chromatin-modifying complexes such as PRC2 (polycomb-repressive complex 2) and LSD1 (lysine-specific demethylase 1) to the target gene promoters leading to their gene silencing. Various studies have demonstrated that HOTAIR overexpression is associated with breast cancer. Recent studies from our laboratory demonstrate that HOTAIR is required for viability of breast cancer cells and is trans...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Expression Profiles of Estrogen-Regulated MicroRNAs in Breast Cancer Cells
Molecular signaling through both estrogen and microRNAs are critical for breast cancer development and growth. The activity of estrogen is mediated by transcription factors, the estrogen receptors. Here we describe a method for robust characterization of estrogen-regulated microRNA profiles. The method details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, microRNA large-scale profiling, and subsequent confirmations. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Competitive Binding Assay for the G-Protein-Coupled Receptor 3 (GPR3) or G-Protein-Coupled Estrogen Receptor (GPER)
The role of 2-methoxyestradiol is becoming a major area of investigation because of its therapeutic utility, though its mechanism is not fully explored. Recent studies have identified the G-protein-coupled receptor 30 (GPR30, GPER) as a high-affinity membrane receptor for 2-methoxyestradiol. However, studies aimed at establishing the binding affinities of steroid compounds for specific targets are difficult, as the tracers are highly lipophilic and often result in nonspecific binding in lipid-rich membrane preparations with low-level target receptor expression. 2-Methoxyestradiol binding studies are essential to elucidate ...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Experimental Procedures for Demonstration of MicroRNA Mediated Enhancement of Functional Neuroprotective Effects of Estrogen Receptor Agonists
This article describes the protocols for the cell viability assay, transfection of VSC4.1 motoneurons with miRs, Annexin V/propidium iodide staining for apoptosis, Western blotting, patch-clamp recording of whole-cell membrane potential, and JC-1 staining for detection of mitochondrial membrane potential. Taken together, these protocols are used to demonstrate that miR-7-1 caused significant enhancement of the efficacy of estrogen receptor agonists for functional neuroprotection in VSC4.1 motoneurons. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Silencing Estrogen Receptor-β with siRNA in Cultured Cells
Estrogen receptors α and β (ERα and ERβ) are the two genomic estrogen receptors. ERβ was the second of these receptors to be discovered; its structure is similar to that of ERα but they are different in histological distribution. However, the functions of ERα versus ERβ are still unclear. The ability of small interfering RNAs (siRNAs) to silence gene expression has proven to be invaluable for studying gene function in cultured mammalian cells. This chapter describes the use of siRNA to inhibit the expression of ERβ in renal cell carcinoma (RCC) and to further the understandi...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Silencing Estrogen Receptor-α with siRNA in the Intact Rodent Brain
The ability to silence the expression of gene products in a chemically, spatially, and temporally specific manner in the brains of animals has enabled key breakthroughs in the field of behavioral neuroscience. Using this technique, estrogen receptor alpha (ERα) has been specifically implicated in a multitude of behaviors in mice, including sexual, aggressive, locomotor, and maternal behaviors. ERα has been identified in a variety of brain regions, including the medial preoptic area, ventromedial hypothalamus, and amygdala. In this chapter we describe the techniques involved in the generation of the small hairpi...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Estrogen Receptor Agonists and Antagonists in the Yeast Estrogen Bioassay
Cell-based bioassays can be used to predict the eventual biological activity of a substance on a living organism. In vitro reporter gene bioassays are based on recombinant vertebrate cell lines or yeast strains and especially the latter are easy-to-handle, cheap, and fast. Moreover, yeast cells do not express estrogen, androgen, progesterone or glucocorticoid receptors, and are thus powerful tools in the development of specific reporter gene systems that are devoid of crosstalk from other hormone pathways. This chapter describes our experience with an in-house developed RIKILT yeast estrogen bioassay for testing estrogen r...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Comparison of the Effects of the Selective Estrogen Receptor Modulators Ospemifene, Raloxifene, and Tamoxifen on Breast Tissue in Ex Vivo Culture
Explant tissue culture provides a model for studying the direct effects of steroid hormones, their analogs, and novel hormonally active compounds on normal freshly isolated human breast tissues (HBTs). For this purpose, pre- and postmenopausal HBTs can be maintained in this culture system. The results demonstrate that the morphological integrity of HBT explants can be maintained in tissue culture up to 2 weeks and expression of differentiation markers, steroid hormone receptors, proliferation and apoptosis ratios can be evaluated as a response to hormonal stimulation. This chapter describes an ex vivo culture model that we...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Use of Reporter Genes to Analyze Estrogen Response: The Transgenic Zebrafish Model
In vivo models to detect estrogenic compounds are very valuable for screening for endocrine disruptors. Here we describe the use of transgenic estrogen reporter zebrafish as an in vivo model for identification of estrogenic properties of compounds. Live imaging of these transgenic fish provides knowledge of estrogen receptor specificity of different ligands as well as dynamics of estrogen signaling. Coupled to image analysis, the model can provide quantitative dose-response information on estrogenic activity of chemical compounds. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
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
Selective estrogen receptor modulators (SERMs) are a class of compounds that act differentially on the estrogen receptor (ER) in various tissues with a mixed agonist/antagonistic activity (agonistic in some tissues while antagonist in others). This peculiarity represents a challenge for developing new hormone replacement therapies (HRTs) and highlights the need of new tools to evaluate the specific effects of a given SERM in different organs/tissues of an entire organism and with time. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
The Synonymous Ala87 Mutation of Estrogen Receptor Alpha Modifies Transcriptional Activation Through Both ERE and AP1 Sites
Estrogen receptor α (ERα) exerts regulatory actions through genomic mechanisms. In the classical pathway, ligand-activated ERα binds directly to DNA through estrogen response elements (ERE) located in the promoter of target genes. ERα can also exert indirect regulation of transcription via protein-protein interaction with other transcription factors such as AP-1. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Tips and Tricks for Successful Application of Statistical Methods to Biological Data
This chapter discusses experimental design and use of statistics to describe characteristics of data (descriptive statistics) and inferential statistics that test the hypothesis posed by the investigator. Inferential statistics, based on probability distributions, depend upon the type and distribution of the data. For data that are continuous, randomly and independently selected, as well as normally distributed more powerful parametric tests such as Student’s t test and analysis of variance (ANOVA) can be used. For non-normally distributed or skewed data, transformation of the data (using logarithms) may normalize th...
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
Surface Plasmon Resonance Study of Cooperative Interactions of Estrogen Receptor α and Specificity Protein 1 with Composite DNA Elements
This study is pivotal in guiding the bioinformatics simulation to yield an exact model of the spacer dependency of the transcription factor/cofactor–DNA interactions, which is important for understanding the nuclear receptor regulating activity through other coactivators. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
The Estrogen Receptors: An Overview from Different Perspectives
The estrogen receptors, ERα, ERβ, and GPER, mediate the effects of estrogenic compounds on their target tissues. Estrogen receptors are located in the tissues of the female reproductive tract and breast as one would expect, but also in tissues as diverse as bone, brain, liver, colon, skin, and salivary gland. The purpose of this discussion of the estrogen receptors is to provide a brief overview of the estrogen receptors and estrogen action from perspectives such as the historical, physiological, pharmacological, pathological, structural, and ligand perspectives. (Source: Springer protocols feed by Biochemistry)
Source: Springer protocols feed by Biochemistry - October 12, 2015 Category: Biochemistry Source Type: news
