细胞技术

The expression of a recombinant MMP in a mammalian cell line can be useful, e.g., for purification of the enzyme, to characterize function of the enzyme, or to uncover its substrates. In this chapter, we have therefore documented our experience with the recently discovered MMP-28.

As their name implies, matrix metalloproteinases (MMPs) are thought to be responsible for the turnover of connective tissue proteins, a function that is indeed performed by some family members. However, matrix degradation is possibly not the predominant function of these enzymes. Seve ...

The “a disintegrin and metalloproteinase with thrombospondin motifs” (ADAMTS) enzymes are secreted proteinases involved in development, blood clotting and the turnover of extracellular matrix. Manufacturing recombinant enzyme presents quite a challenge due to the presen ...

Matrix metalloproteinases (MMPs) modulate cell–cell and cell–matrix interactions and the membrane-type MMPs (MT-MMPs) are especially important for pericellular proteolysis. Understanding the activity, regulation, and function of MT-MMPs would provide important insi ...

Signal Transducer and Activator of Transcription (STAT) proteins are latent cytoplasmic transcription �factors that become activated by phosphorylation at a C-terminal tyrosine residue. Upon activation STAT proteins translocate to the nucleus and bind to their specific tar ...

JAK kinases are critical mediators in development, differentiation, and homeostasis and accordingly, have become well-validated targets for drug discovery efforts. In recent years, the integration of X-ray crystallography in kinase-focused drug discovery programs has prov ...

The eight SOCS (Suppressor of Cytokine Signaling) proteins encoded in the human genome all contain a C-terminal domain, the SOCS box, that allows them to function as E3 ubiquitin ligases and thereby catalyze the ubiquitination of components of the JAK/STAT signaling pathway. This activity is ...

The suppressors of cytokine signalling (SOCS) protein family consist of eight members (SOCS 1–7, and CIS). SOCS1 and SOCS3 are the best-studied family members and have been shown to act as negative feedback inhibitors of the JAK/STAT signalling pathway. To study the physiological roles of the SO ...

The Suppressor of Cytokine Signalling (SOCS) proteins are a family of negative regulators characterized by a central SH2 domain and C-terminal SOCS box motif. Cytokine Inducible SH2-containing protein (CIS), SOCS1, 2 and 3 are rapidly upregulated in response to cytokine stimulation and ...

STAT proteins are activated by diverse cellular stimuli including cytokine and growth factor receptor signaling, proto-oncogene and oncogene expression, and cellular stress mediators. In most cases, canonical STAT activation by a particular treatment or cellular condition r ...

The transcription factor Signal Tranducer and Activator of Transcription 5 (STAT5) plays an important role in many biological processes. To study STAT5 biology, several different constructs have been designed that render STAT5 constitutively active. These constructs have now be ...

Multiple experimental tools have demonstrated that cytokine-induced STAT activation entails the transition of dimer conformations rather than de novo dimerization. In this chapter, we describe the utilization of analytical ultracentrifugation (AUC) as a powerful techniq ...

The ability to observe the dynamic localization of a protein in living cells can provide critical insight to its mode of action and functional molecular interactions. To this purpose, green fluorescent protein (GFP) has served as a powerful tool to tag STAT proteins for microscopic visualiz ...

Phosphorylation of signal transducers and activators of transcription (STATs) indicates their involvement in active signaling. Here we describe immunohistochemical staining procedures for detection and identification of the cellular localization of phospho-STAT2 ...

Flow cytometry can be used to study STAT phosphorylation on a per cell basis. Cells are fixed, permeablized, and stained with antibodies that specifically bind to the phosphorylated form of the STAT protein. This allows the tyrosine phosphorylation of a single STAT to be studied within a heterog ...

Acetylation of signal transducer and activator of transcription (STAT) proteins has been recognized as a significant mechanism for the regulation of their cellular functions. Site-specific antibodies are available only for a minority of STATs. The detection of acetylated STATs by i ...

Here we describe the preparation of nuclear extracts and the electrophoretic mobility shift assay (EMSA) for the detection of STAT species. We use the method for the investigation of STAT1 and STAT3 homo- and heterodimers and show how the preparation of the extracts can influence the distribu ...

Mutations in the JAK2 gene are prevalent in the human myeloid malignancies, being present in virtually all cases of polycythemia vera, and a significant proportion of patients with other myeloproliferative disorders. Various methods for the detection of acquired mutations in this ge ...

The JAK family of protein tyrosine kinases are now recognized as important participants in a wide range of pathologies, from cancer to inflammatory diseases. In the last decade, the drive to develop drugs targeting members of this family has begun to deliver a panel of small molecule inhibitors of ...

The JAK/STAT signaling pathway has essential roles in multiple developmental processes, including stem cell maintenance, immune responses, and cellular proliferation. As a result, it has been extensively studied in both vertebrate systems and lower complexity models, such as Dro ...