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Using the MEROPS Database for Proteolytic Enzymes and Their Inhibitors and Substrates
Neil D. Rawlings, Alan J. Barrett, Alex Bateman

MEROPS is a database of proteolytic enzymes as well as their inhibitors and substrates. Proteolytic enzymes and protein inhibitors are organized into protein domain families. In turn, families are organized into clans.

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Using the Structure‐Function Linkage Database to Characterize Functional Domains in Enzymes
Shoshana Brown, Patricia Babbitt

The Structure‐Function Linkage Database (SFLD; http://sfld.rbvi.ucsf.edu/) is a Web‐accessible database designed to link enzyme sequence, structure, and functional information.

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Using EMBL‐EBI Services via Web Interface and Programmatically via Web Services
Rodrigo Lopez, Andrew Cowley, Weizhong Li, Hamish McWilliam

The European Bioinformatics Institute (EMBL‐EBI) provides access to a wide range of databases and analysis tools that are of key importance in bioinformatics.

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Clustal Omega
Fabian Sievers, Desmond G. Higgins

Clustal Omega is a package for making multiple sequence alignments of amino acid or nucleotide sequences, quickly and accurately. It is a complete upgrade and rewrite of earlier Clustal programs.

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Genome Annotation and Curation Using MAKER and MAKER‐P
Michael S. Campbell, Carson Holt, Barry Moore, Mark Yandell

This unit describes how to use the genome annotation and curation tools MAKER and MAKER‐P to annotate protein‐coding and noncoding RNA genes in newly assembled genomes, update/combine legacy annotations in light of new evidence, add quality metrics to annotations from other pipelines, and m.

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DXMSMS Match Program for Automated Analysis of LC‐MS/MS Data Obtained Using Isotopically Coded CID‐Cleavable Cross‐Linking Reagents
Evgeniy V. Petrotchenko, Karl A.T. Makepeace, Christoph H. Borchers

Cross‐linking combined with mass spectrometry for the study of proteins and protein complexes is greatly facilitated by the use of isotopically coded cleavable cross‐linking reagents.

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Standardized Isolation and Culture of Murine Liver Sinusoidal Endothelial Cells
Rajkumar Cheluvappa

Isolation of murine liver sinusoidal endothelial cells (LSECs) is an exacting and finicky procedure. After exhaustive standardization, we were able to devise an easily reproducible protocol which produced consistent results.

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Freeze‐Fracture Immunocytochemistry: Fracture‐Label and Label‐Fracture for the Localization of Membrane Proteins
Frederick W. K. Kan

Freeze‐fracture is a unique investigative tool for visualization of the en face topography of individual membrane leaflets of cell membranes at high resolution under the electron microscope.

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In Situ Detection of Integrin Ligands
Daiji Kiyozumi, Ryoko Sato‐Nishiuchi, Kiyotoshi Sekiguchi

Integrins are cell surface receptors for cell adhesion. Integrin‐mediated cell adhesion regulates various cellular processes, including cell survival, migration, proliferation, and differentiation.

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In Situ Detection of Integrin Ligands
Daiji Kiyozumi, Ryoko Sato‐Nishiuchi, Kiyotoshi Sekiguchi

Integrins are cell surface receptors for cell adhesion. Integrin‐mediated cell adhesion regulates various cellular processes, including cell survival, migration, proliferation, and differentiation.

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A High‐Content Assay for Biosensor Validation and for Examining Stimuli that Affect Biosensor Activity
Scott D. Slattery, Klaus M. Hahn

Biosensors are valuable tools used to monitor many different protein behaviors in vivo. Demand for new biosensors is high, but their development and characterization can be difficult.

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Analysis of Copy‐Number Alterations in Single Cells Using Microarray‐Based Comparative Genomic Hybridization (aCGH)
Birte Möhlendick, Nikolas H. Stoecklein

In this unit, we describe a workflow that enables array comparative genomic hybridization (aCGH) of single cells. The unit first describes the isolation and preparation of single peripheral mononuclear cells from blood (PBMC) to prepare a suitable reference DNA for aCGH experiments.

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Mapping Replication Origin Sequences in Eukaryotic Chromosomes
Haiqing Fu, Emilie Besnard, Romain Desprat, Michael Ryan, Malik Kahli, Jean‐Marc Lemaitre, Mirit I. Aladjem

Recent advances in genome‐sequencing technology have led to the complete mapping of DNA replication initiation sites in the human genome.

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Probing Small‐Molecule Microarrays with Tagged Proteins in Cell Lysates
Marius S. Pop, Dina Wassaf, Angela N. Koehler

The technique of small‐molecule microarray (SMM) screening is based on the ability of small molecules to bind to various soluble proteins.

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Determination of the Cytostatic and Cytocidal Activities of Antimalarial Compounds and Their Combination Interactions
Katy S. Sherlach, Paul D. Roepe

Determining the antiplasmodial activity of candidate antimalarial drugs in vitro identifies new therapies for drug‐resistant malaria. Importantly though, activity can be either growth‐inhibitory (cytostatic) or parasite‐kill (cytocidal), or both.

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Real‐Time Chemiluminescence Imaging Using Nano‐Lantern Probes
Yoshiyuki Arai, Takeharu Nagai

Chemiluminescence imaging can be performed without excitation light sources at various spatial levels ranging from a single cell to the whole body.

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Inducible Models of Bone Loss
Casey R. Doucette, Clifford J. Rosen

Bone is an essential organ that not only confers structural stability to the organism, but also serves as a reservoir for hematopoietic elements and is thought to affect systemic homeostasis through the release of endocrine factors as well as calcium.

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Conservation of Mouse Models Through Embryo Freezing
Mo Guan, Debora Bogani, Susan Marschall, Marcello Raspa, Toru Takeo, Naomi Nakagata, Martin Fray

The ability to interrogate the entire coding sequence of the mouse combined with the tools to manipulate the genome has firmly established the mouse as the model organism of choice for studying the causes of human disease.

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Histopathological Analysis of the Respiratory Tract
Cheryl L. Scudamore, Elizabeth F. McInnes

The basic anatomy of the mouse respiratory system is similar to that of other mammals and can be usefully examined under the light microscope in phenotyping studies, inhalation toxicity studies, and studies involving mouse models of human disease.

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Ferric Chloride–Induced Arterial Thrombosis in Mice
Saravanan Subramaniam, Sandip M. Kanse

Because of its high sensitivity and reproducibility, the FeCl3‐induced arterial thrombosis mouse model is widely used to study molecular mechanisms as well as the efficacy of antithrombotic agents.

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Serial Block Face‐Scanning Electron Microscopy: A Method to Study Retinal Degenerative Phenotypes
Debarshi Mustafi, Sandra Kikano, Krzysztof Palczewski

Retinal degenerative conditions can vary in their clinical features and often present with subtle phenotypic features before the onset of clinically overt disease.

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NMR Analysis of Base‐Pair Opening Kinetics in DNA
Marta W. Szulik, Markus Voehler, Michael P. Stone

Base pairing in nucleic acids plays a crucial role in their structure and function.

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Steady‐State Kinetic Analysis of DNA Polymerase Single‐Nucleotide Incorporation Products
Derek K. O'Flaherty, F. Peter Guengerich

This unit describes the experimental procedures for the steady‐state kinetic analysis of DNA synthesis across DNA nucleotides (native or modified) by DNA polymerases.

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Detection of Hydrogen Bonds in Dynamic Regions of RNA by NMR Spectroscopy
Andre Dallmann, Michael Sattler

NMR spectroscopy is a powerful tool to study the structure and dynamics of nucleic acids.

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Olefin Cross‐Metathesis for the Synthesis of Alkenyl Acyclonucleoside Phosphonates
Maxime Bessières, Coralie De Schutter, Vincent Roy, Luigi A. Agofoglio

The detailed synthetic protocol for the straightforward, efficient synthesis of various alkenyl acyclonucleosides, including challenging trisubstituted alkenyl acyclonucleoside phosphonates, is described.

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Practical Synthesis of 4′‐Thioribonucleosides Starting from D‐Ribose
Noriaki Minakawa, Akira Matsuda

A practical synthesis of 4′‐thioribonucleosides, i.e., 4′‐thiouridine, ‐cytidine, ‐adenosine, and ‐guanosine, which are versatile units for nucleic acids–based therapeutics, is described.

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Overview of Different Mechanisms of Arrestin‐Mediated Signaling
Vsevolod V. Gurevich, Eugenia V. Gurevich

Arrestins are characterized by their ability to selectively bind active, phosphorylated GPCRs and suppress (arrest) receptor coupling to G proteins. Nonvisual arrestins are also signaling proteins in their own right, activating a variety of cellular pathways.

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Arrestin Expression in E. coli and Purification
Sergey A. Vishnivetskiy, Xuanzhi Zhan, Qiuyan Chen, Tina M. Iverson, Vsevolod V. Gurevich

Purified arrestin proteins are necessary for biochemical, biophysical, and crystallographic studies of these versatile regulators of cell signaling. Described herein is a basic protocol for arrestin expression in E.

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Guinea Pig Models of Asthma
Alice E. McGovern, Stuart B. Mazzone

Described in this unit are methods for establishing guinea pig models of asthma. Sufficient detail is provided to enable investigators to study bronchoconstriction, cough, airway hyperresponsiveness, inflammation, and remodeling. © 2014 by John Wiley & Sons, Inc.

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Modeling Human Liver Cancer Heterogeneity: Virally Induced Transgenic Models and Mouse Genetic Models of Chronic Liver Inflammation
Marc Ringelhan, Florian Reisinger, Detian Yuan, Achim Weber, Mathias Heikenwalder

In addition to being the most common primary liver cancer, hepatocellular carcinoma (HCC) is the second most common cause of cancer‐related death in humans.

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