细胞功能测定

The yeast two-hybrid system is a sensitive method to detect intracellular interactions between proteins. It has been successfully employed in the identification of novel interactors of several proteins, including adhesion molecules (for a general review on the method, see refs. 1 and 2). ...

Several antireceptor monoclonal antibodies (MAbs) have been described to compete with ligands for receptor binding. The possibility that structures derived from the hypervariable or complementarity determining regions (CDR) of such antibodies display similarity with th ...

This chapter will describe two approaches that utilize the polymerase chain reaction (PCR) to clone new members of previously known adhesion receptor families. These two approaches are useful to clone either alternatively spliced forms of previously known family members or novel mol ...

Screening cDNA libraries by transient expression in mammalian cells has proven to be very effective for the isolation of cDNAs encoding secreted, surface, and intracellular proteins.

Production of monoclonal antibodies (MAbs) using adhesion-involved cell types as a source of antigens allows the identification of new molecules participating in the adhesion process (1–3). Immunoaffinity purification followed by protein microsequencing is one of the techni ...

Over the past few years, soluble forms of adhesion molecules and cell-surface proteins in general have become widely used tools, not only in the study of protein-protein interactions, but also as affinity probes to identify novel ligands for a given cell-surface molecule. Particularly use ...

The development and the extensive use of immunofluorescence microscopy on cultured cells has greatly improved the knowledge of cell structure. Immunofluorescence microscopy has become a major tool that any cell and molecular biologist cannot afford to ignore.

Immunohistochemistry allows detection of antigenic molecules in situ in tissue samples using a standard light microscope. This technique has been used widely in recent years in diagnostic pathology and in research. The large popularity of immunohistochemistry came after the disc ...

Various assays have been employed to study the nuclear receptor/cofactor interactions. Coimmunopre-cipitation protocols, both yeast and mammalian two-hybrid systems, and electrophoretic mobility shift/ supershift assays are all commonly used. One of the most useful assays f ...

Steroid hormone receptor-mediated reporter assays in the budding yeast Saccharomyces cerevisiae have been an invaluable tool for the identification and functional characterization of steroid hormone receptor-associated chaperones and cochaperones. This chapter de ...

The identification of direct nuclear hormone receptor gene targets provides clues to their contribution to both development and cancer progression. Until recently, the identification of such direct target genes has relied on a combination of expression analysis and in silico searc ...

Defining the precise promoter DNA sequence motifs where nuclear receptors and other transcription factors bind is an essential prerequisite for understanding how these proteins modulate the expression of their specific target genes. The purpose of this chapter is to provide the rea ...

Quantitative imaging techniques of fluorescently-tagged proteins have been instrumental in the study of the behavior of nuclear receptors (NRs) and coregulators in living cells. Ligand-activated NRs exert their function in transcription regulation by binding to specific res ...

Crystallographic analysis of the ligand-binding domains of nuclear hormone receptors (NR) has provided a unique insight into the molecular events that underlie the ligand-mediated control of their transcriptional activity. The technique relies on preparing milligram quant ...

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 ...