细胞技术

Measurement of protein expression in live, intact cells using flow cytometry (FC) has been employed for several decades in the areas of immunology, cell biology, and molecular biology. More recently, this technique has found appreciation in applied scientific fields, including cancer ...

Understanding the molecular mechanisms of steroid hormone action requires assays that measure rates of ligand dissociation and receptor degradation. Ligand dissociation is a pseudo-first order reaction of a high affinity -labeled ligand. Receptor turnover as described here is ...

Genetic alterations underlying the development of cancer include large chromosomal aberrations, such as amplifications, deletions and translocations as well as small changes in sequence, i.e. mutations. Thus, different methods are needed to reveal various types of genetic chan ...

The use of tissue-selective rather than ubiquitous knockouts of steroid receptors allows a more refined study of the mechanism of steroid action in defined target tissues and circumvents problems such as early lethality or major developmental defects precluding studies in affected ...

Protein phosphorylation is a versatile posttranslational modification that can regulate nuclear receptor function. Although the precise role of receptor phosphorylation is not fully understood, it appears that it functions to direct or refine receptor activity in response to p ...

X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have proved powerful methods for studying the structure of the isolated ligand and DNA-binding domains of nuclear receptors. However, the N-terminal domain (NTD), which in some members of the superfamily is imp ...

RNA interference can be extremely useful in determining the function of an endogenously-expressed protein in its normal cellular environment. In this chapter, we describe a method that uses small interfering RNA (siRNA) to knock down mRNA and protein expression in cultured cells so that the ...

Knowledge of the kinetics of protein—protein interactions has become important in defining nuclear receptor function. Such knowledge allows characterization of interactions that occur with high affinity and/or selectivity. Surface plasmon resonance is a useful and sensiti ...

It is just over 20 years since the first steroid receptor cDNAs were cloned, a development that led to the birth of a superfamily of ligand activated transcription factors: the nuclear receptors. Natural ligands for nuclear receptors are generally lipophilic in nature and include steroid ho ...

The methods described are designed to enable the assignment of an intracellular localization of secretory proteins, either soluble or membrane associated, to later secretory compartments, such as the trans-Golgi network (TGN) or endosome. These two subcellular compartments are c ...

Sphingolipids are constituents of biological membranes. Ceramide and sphingosine 1-phosphate (S1P) also act as second messengers and are part of a rheostat system, in which ceramide promotes cell death and growth arrest, and S1P induces proliferation and maintains cell survival. As ma ...

Macroautophagy (herein referred to as autophagy) contributes to the control of life and death throughout the animal and plant kingdoms. Bilateral links have been found between apoptosis and autophagy where inducers of apoptosis also induce autophagy and vice versa. In some cases, autop ...

The accumulation of mutant aggregate-prone proteins is a feature of several human disorders, collectively referred to as protein conformation disorders or proteinopathies. We have shown that autophagy, a cytosolic, non-specific bulk degradation system, is an important cleara ...

Intracellular antigens can be presented on major histocompatibility complex (MHC) class II molecules after degradation via macroautophagy. To enhance MHC class II presentation of potential vaccine antigens, we have developed a method to target antigens for autophagic degrada ...

Chaperone-mediated autophagy (CMA) is the only type of autophagy in mammalian cells able to selectively degrade cytosolic proteins in lysosomes. CMA is maximally activated in response to stressors such as prolonged starvation, exposure to toxic compounds, or oxidative stress. We have ...

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar mem ...

The ability to accurately monitor the dynamics involved with the activity and state of a specific protein population in a complex biological system represents one of the major technological challenges in studying systems biology. Over the past several years a number of groups have attemp ...

Recent studies indicate that protein glutathionylation is an important regulatory mechanism. The develop-ment of redox proteomics techniques to identify proteins undergoing glutathionylation has a key role in defining the importance of this post-translational modific ...

Protein phosphorylation is central to most signaling events in eukaryotic cells. Large-scale analysis of protein phosphorylation in vivo is a highly challenging undertaking that requires powerful analytical and bioinformatics tools; numerous phosphoproteomic method ...

Ubiquitination involves the tagging of proteins with one (mono-) or more (poly-) ubiquitin molecules. Primarily the role of ubiquitination involves mainly short-lived and regulatory proteins being tagged with a poly-ubiquitin tail, thus introducing a hydrophobic patch that allo ...