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Biochemistry

Individual-nucleotide-resolution UV Cross-linking and Immunoprecipitation (iCLIP) of UPF1 Authors:  David Zünd and Oliver Mühlemann, date: 04/05/2014, view: 8073, Q&A: 0
The fate of mRNA, in particular its stability, localization and rate of translation is regulated by RNA binding proteins assembling to messenger ribonucleoprotein (mRNP) complexes. To investigate the transcriptome-wide RNA binding sites of UPF1, the core factor of nonsense-mediated mRNA decay (NMD), we performed individual-nucleotide-resolution UV ...
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UPF1 RNA Immunoprecipitation from Mini-μ Construct–expressing Cells Authors:  David Zünd and Oliver Mühlemann, date: 04/05/2014, view: 5206, Q&A: 0
UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally ...
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Cancer Biology

Isolation and Immortalization of Fibroblasts from Different Tumoral Stages Authors:  Fernando Calvo, Steven Hooper and Erik Sahai, date: 04/05/2014, view: 9343, Q&A: 0
Tumour microenvironment and cancer-associated fibroblasts in particular exhibit tumour promoting abilities that are not present in their normal counterparts (Calvo et al., 2013; Hanahan and Coussens, 2012). Therefore, functional and molecular characterization of the modifications occurring in fibroblasts during tumour progression is ...
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Immunology

α2β1-integrin Clustering and Internalization Protocol Authors:  Varpu Marjomäki, Mikko Karjalainen, Paula Upla, Elina Siljamäki, Nina Rintanen and Paula Turkki, date: 04/05/2014, view: 4354, Q&A: 0
α2β1-integrin clustering experiment can be used to trigger internalization of α2β1-integrin. When clustering is performed with sequential administration of primary and fluorescent secondary antibodies, the entry kinetics of integrin can be followed into the cell. The idea is first to allow binding of primary antibodies (recognizing the ...
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Isolation of Cells from Human Intestinal Tissue Authors:  Heli Uronen-Hansson, Emma Persson, Petra Nilsson and William Agace, date: 04/05/2014, view: 8897, Q&A: 0
The intestinal lamina propria contains a dense network of T cells, dendritic cells (DCs) and macrophages, which play an important role in local innate and adaptive immune responses. We have recently identified distinct subsets of DCs (Persson et al., 2013) and macrophages (Bain et al., 2013) in the human intestine. In addition, ...
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Microbiology

Fluorescence Microscopy Analysis of Drug Effect on Autophagosome Formation Authors:  Metodi Stankov, Diana Panayotova-Dimitrova, Martin Leverkus and Georg Behrens, date: 04/05/2014, view: 5752, Q&A: 0
The autophagy protein, LC3 represents a reliable characteristic marker for autophagosomal structures. The initial LC3 is processed by the cysteine protease autophagy-related gene 4 (Atg4) at its C terminus in order to create LC3-I generally localized in the cytoplasm. Afterwards LC3-I is conjugated with phosphatidylethanolamine (PE) to become ...
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Flow Cytometric Analysis of Autophagic Activity with Cyto-ID Staining in Primary Cells Authors:  Metodi Stankov, Diana Panayotova-Dimitrova, Martin Leverkus, Jan-Henning Klusmann and Georg Behrens, date: 04/05/2014, view: 12537, Q&A: 0
Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the ...
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Flow Cytometric Analyses of Autophagic Activity using LC3-GFP fluorescence Authors:  Metodi Stankov, Diana Panayotova-Dimitrova, Martin Leverkus and Georg Behrens, date: 04/05/2014, view: 7235, Q&A: 0
Flow cytometry allows very sensitive and reliable high-throughput analysis of autophagic flux. This methodology permits to screen cells in flow and capture multi-component images. Using this technology autophagic flux may be analysed accurately in both suspension as well as adherent cells upon trypsinization independent of how heterogeneous the ...
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Native BAD-1 Binding to Heparin-agarose Author:  T. Tristan Brandhorst, date: 04/05/2014, view: 3354, Q&A: 0
BAD-1 is an adhesin created by the dimorphic fungus Blastomyces dermatitidis, the causative agent of blastomycosis. We have determined that it has an affinity for heparin, which may explain its impact on virulence and human immune function as a number of cells related to immune function have heparin like moieties on their surfaces. This ...
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Molecular Biology

Electrophoresis Mobility Shift Assay Authors:  Masaru Nakata and Masaru Ohme-Takagi, date: 04/05/2014, view: 7805, Q&A: 0
Protein (transcription factors and/or transcription cofactors)-binding to DNA is a critical event in regulation of transcription. Electrophoresis Mobility Shift Assay (EMSA), also known as gel shift assay, is a useful tool to detect protein- or protein complex-DNA/RNA interaction and to evaluate DNA binding specificity of transcription factors in ...
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Plant Science

DNA Damage Sensitivity Assays with Arabidopsis Seedlings Authors:  Marisa Rosa and Ortrun Mittelsten Scheid, date: 04/05/2014, view: 5384, Q&A: 0
We describe fast and reproducible sensitivity assays to quantify the response of Arabidopsis seedlings of different genotypes to a wide range of DNA damaging agents. We apply (1) γ-irradiation, which produces DNA breaks, (2) bleocin, a radiomimetic drug, (3) mitomycin C, a DNA intrastrand cross-linker, (4) hydroxyurea, an inhibitor of DNA ...
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Measuring Homologous Recombination Frequency in Arabidopsis Seedlings Authors:  Marisa Rosa and Ortrun Mittelsten Scheid, date: 04/05/2014, view: 6174, Q&A: 0
Somatic homologous recombination (SHR) is a major pathway of DNA double-strand break (DSB) repair, in which intact homologous regions are used as a template for the removal of lesions. Its frequency in plants is generally low, as most DSB are removed by non-homologous mechanisms in higher eukaryotes. Nevertheless, SHR frequency has been shown to ...
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Seed Coat Ruthenium Red Staining Assay Authors:  Heather E. McFarlane, Delphine Gendre and Tamara L. Western, date: 04/05/2014, view: 9252, Q&A: 0
The goal of this protocol is to assay for defects in synthesis/secretion/release of seed coat mucilage by ruthenium red staining of mature whole seeds.

The mucilage secretory cells of the Arabidopsis seed coat synthesize and secrete a large quantity of primarily pectinaceous mucilage to a ring-shaped apical domain during their ...
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Quantification of Anthocyanin Content Authors:  Masaru Nakata and Masaru Ohme-Takagi, date: 04/05/2014, view: 12232, Q&A: 1
Anthocyanins are a class of flavonoids and important plant pigments. They attract insects to pollinate flowers, protect plants from UV irradiation, and act as antimicrobial agents against herbivores and pathogens. Biosynthesis of anthocyanin is stimulated by diverse developmental signals and environmental stresses including drought, wounding, ...
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Stem Cell

3D Mammary Colony-Forming Cell Assay Authors:  Giusy Tornillo and Sara Cabodi, date: 04/05/2014, view: 8597, Q&A: 0
The mammary epithelium consists of multiple phenotypically and functionally distinct cell populations, which are organized as a hierarchy of stem cells, progenitors and terminally differentiated cells.

Identification of the mechanisms regulating the growth and differentiation of mammary stem and progenitor cells is of great interest ...
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