细胞技术

The Rit subfamily of GTPases is a founding branch within the Ras family of small G-proteins and preserves unique sequences in the G2 effector loop domain and the C-terminus. Rit proteins regulate a diversity of signal transduction pathways, some of which are similar to and others of which differ from ...

The in vitro kinase assay is an invaluable method to identify and characterize kinase substrates and to determine specific site(s) of phosphorylation. However, the use of recombinant proteins does not adequately account for the contribution of accessory proteins, such as scaffolds, th ...

The Ras-related GTPase Rap has been implicated in multiple cellular functions. In particular, Rap1 is a crucial regulator of both inside-out integrin activation and outside-in E-cadherin-mediated signaling. Thus, Rap1 was proposed as a fundamental regulator of the cross talk between ...

The coordinate modulation of the cellular functions of cadherins and integrins plays an essential role in fundamental physiological and pathological processes, including morphogenesis, tissue differentiation and renewal, wound healing, immune surveillance, inflamm ...

Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a c ...

Identification of oncogene-mediated phosphorylation events is essential to understanding the molecular determinants responsible for cancer development and progression. Here, we identify KRAS-regulated phosphorylation events using label-free quantitation-b ...

Chromatin packaging plays a significant role in regulating gene transcription. Study of the higher-order packing states of chromatin by image analysis at the light microscope level, especially when validated by methods of molecular biology, immunochemistry, and/or immunocyto ...

The thymidine analogue 5-bromo-2′-deoxyuridine (BrdU) has been widely used to make sister chromatid differentiation (SCD) evident in metaphase chromosomes of cells grown for two cycles in BrdU and, thus, containing varying amounts of the thymidine analogue. A direct consequence was t ...

DNA labeling in vivo using nucleoside analogues is a current experimental approach to determine cell proliferation rates in cell cultures and tissues. It has also been successfully used to localize adult stem cell niches through the identification of nucleoside label-retaining cel ...

Analysis of the formation of extended chromatin fibers (ECFs) in response to the action of gravity following lysis by hypertonic and detergent solutions is a useful technical procedure relevant for studies of the positioning of particular DNA signals on chromatin filaments. Addition ...

Over the past three decades the development of methods for visualizing at the cell level the extent of DNA breakage significantly contributed to genotoxicity testing: their availability greatly improved the knowledge in the field of genetic toxicology. These procedures are based on the ...

A useful tool in the detection of overall and region-specific DNA damage is the Comet-FISH technique. This method combines two well-established methods, the Comet assay (single cell gel electrophoresis), which makes it possible to detect and quantify DNA damage at the single cell level, and FI ...

Mixtures of polychrome methylene blue-eosin Y (i.e., Giemsa stain) are widely used in biological staining. They induce a striking purple coloration of chromatin DNA (the Romanowsky-Giemsa effect), which contrasts with the blue-stained RNA-containing cytoplasm and nucleoli. After ...

Binding of small molecules with DNA plays an important role in many biological functions such as DNA replication, repair, and transcription. These interactions also offer enormous potential as targets for diagnostics and therapeutics, leading to intense interest in development of m ...

Chromatin-associated RNA (caRNA) is a newly identified class of RNA stably linked to chromatin and responsible for maintaining the higher order structure of euchromatic regions in an accessible form (Schubert et al., Mol Cell 48, 434 – 444, 2012). This section provides a pipeline of bioinforma ...

Chromatin-associated RNA (caRNA) is a newly identified class of RNA molecules stably associated with chromatin, maintaining the higher order structure of euchromatic regions in an accessible form (Schubert et al., Mol Cell, doi:10.1016/j.molcel.2012.08.021, 2012). This section pr ...

Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a l ...

Chromatin immunoprecipitation (ChIP) is a powerful method that allows to probe specific protein-DNA interactions in vivo and to estimate the occupancy of proteins at specific sites of the genome. However, the traditional ChIP assay is not able to distinguish whether repeats that share id ...

Chromatin plays important functions in regulating many biological processes, including DNA transcription, replication, and repair. The use of chromatin immunoprecipitation (ChIP) assays has contributed enormously to identify interactions between DNA and a wide range of nu ...

The histones are essential basic proteins intimately involved in most DNA-templated processes. Thus, their purification and fractionation for analysis and their use for in vitro chromatin transactions are of fundamental importance for understanding their role in chromatin st ...