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细胞功能测定

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Multicolor BiFC Analysis of G Protein Complex Formation and Localization

Cells co-express multiple G protein β and γ subunit isoforms, but the extent to which individual subunits associate to form particular βγ complexes is not known. This issue is important because in vivo knockout experiments suggest that specific βγ complexes may have unique functions despi ...

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Using Quantitative BRET to Assess G Protein-Coupled Receptor Homo- and Heterodimerization

Over a period of 15 years the concept of G protein-coupled receptor (GPCR) dimerization moved from a challenging hypothesis to a scientific fact, which is now accepted by the vast majority of the scientists working in the field. However, several important issues remain debated such as the biolog ...

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Reconstitution of G Protein-Coupled Receptors into a Model Bilayer System: Reconstituted High-Density Lipoprotein Particles

Reconstituted high-density lipoprotein particles (rHDL) are powerful platforms used as a model phospholipid bilayer system to study membrane proteins. They consist of a discoidal-shaped planar bilayer of phospholipids that is surrounded by a dimer of apolipoprotein A-I (apoA-I). ...

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Analysis of GPCR/Ion Channel Interactions

Voltage-gated calcium channels are key regulators of calcium homeostasis in excitable cells. A number of cellular signaling pathways serve to fine tune calcium channel activity, including the activation of G protein-coupled receptors. Besides regulating channel activity via s ...

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Cell-Surface ProteinProtein Interaction Analysis with Time-Resolved FRET and Snap-Tag Technologies: Application to G Protein-Coupled Receptor Oligomer

G protein-coupled receptors (GPCRs) are key players in cell–cell communication, the dysregulation of which has often deleterious effects leading to pathologies such as psychiatric and neurological diseases. Consequently, GPCRs represent excellent drug targets, and as such are ...

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Using BRET to Detect Ligand-Specific Conformational Changes in Preformed Signalling Complexes

Bioluminescence energy transfer (BRET) has become a powerful tool to study protein–protein interactions and conformational changes among interacting proteins. In particular, BRET assays performed in living cells have revealed that heptahelical receptors (7TMRs), hetero ...

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Studying Ligand Efficacy at G Protein-Coupled Receptors Using FRET

Drug “ligands” that bind G protein-coupled receptors (GPCRs) can either stimulate, fully (full agonists) or partially (partial agonists), or reduce (inverse agonists) basal receptor activity, by stabilizing different receptor conformations. The term “intrinsic efficacy” was ...

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Bioinformatic Approaches to Metabolic Pathways Analysis

The growth and development in the last decade of accurate and reliable mass data collection techniques has greatly enhanced our comprehension of cell signaling networks and pathways. At the same time however, these technological advances have also increased the difficulty of satisfa ...

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RGS-Insensitive G Subunits: Probes of G Subtype-Selective Signaling and Physiological Functions of RGS Proteins

The Regulator of G protein Signaling (RGS) proteins were identified as a family in 1996 and humans have more than 30 such proteins. Their best known function is to suppress G Protein-Coupled Receptors (GPCR) signaling by increasing the rate of Gα turnoff through stimulation of GTPase activity (i ...

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Improving Drug Discovery with Contextual Assays and Cellular Systems Analysis

Despite rapid growth in our knowledge of potential disease targets following completion of the first drafts of the human genome over 10 years ago, the success rate of new therapeutic discovery has been frustratingly low. In addition to the widely reported costs and single-digit success rate of ...

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Imaging-Based Approaches to Understanding G Protein-Coupled Receptor Signalling Complexes

In the last 10 years, imaging assays based on resonance energy transfer (RET) and protein fragment complementation have made it possible to study interactions between components of G protein-coupled receptor (GPCR) signalling complexes in living cells under physiological condit ...

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Analyzing mRNA Localization to the Endoplasmic Reticulum via Cell Fractionation

The partitioning of secretory and membrane protein-encoding mRNAs to the endoplasmic reticulum (ER), and their translation on ER-associated ribosomes, governs access to the secretory/exocytic pathways of the cell. As mRNAs encoding secretory and membrane proteins comprise ap ...

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Genome-Wide Analysis of RNA Extracted from Isolated Mitochondria

Isolating mitochondria by subcellular fractionation is a well-established method for retrieving intact and functional mitochondria. This procedure has been used to identify proteins of the mitochondria and to explore import mechanisms. Using the same method, it was shown that mit ...

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Using the mRNA-MS2/MS2CP-FP System to Study mRNA Transport During Drosophila Oogenesis

Asymmetric mRNA localisation to specific compartments of the cell is a fundamental mechanism of �spatial and temporal regulation of gene expression. It is used by a variety of organisms and cell types to achieve different cellular functions. However, the mechanisms of mRNA localisation ...

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Imaging mRNAs in Living Mammalian Cells

The gene expression pathway begins in the nucleus as a gene receives a cue to transcribe, and typically ends in the cytoplasm with the production of the required protein. The nuclear processes of mRNA transcription and nucleo-cytoplasmic transport are of high importance as they encompass the ...

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Visualizing Endogenous mRNAs in Living Yeast Using m-TAG, a PCR-Based RNA Aptamer Integration Method, and Fluorescence Microscopy

Localized mRNA translation is involved in cell-fate determination, polarization, and morphogenesis in eukaryotes. While various tools are available to examine mRNA localization, no easy and quick method has allowed for the visualization of endogenously expressed mRNAs in vivo. We ...

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In Vivo Visualization of RNA Using the U1A-Based Tagged RNA System

mRNA transport is a widely used method to achieve the asymmetric distribution of proteins within a cell or organism. In order to understand how RNA is transported, it is essential to utilize a system that can readily detect RNA movement in live cells. The tagged RNA system has recently emerged as a feasi ...

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Visualizing mRNAs in Fixed and Living Yeast Cells

Localization of messenger RNA (mRNA) is a process used by eukaryotes to control the spatio-temporal expression of proteins involved in cellular motility, asymmetric cell division, or polarized cell growth. A better understanding of this process relies on methods to detect specifica ...

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RNA Purification from Tumor Cell Protrusions Using Porous Polycarbonate Filters

Actin-rich cellular protrusions or pseudopodia form via local actin filament polymerization and branching and represent a variety of polarized cellular domains including lamellipodia, filipodia, and neuronal growth cones. RNA localization and local protein translation in ...

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Profiling Axonal mRNA Transport

The importance of mRNA localization and localized protein synthesis to spatially modulate protein levels in distinct subcellular domains has increasingly been recognized in recent years. Axonal and dendritic processes of neurons represent separate functional domains of the ...

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