Nuclear translocation assay

However, some receptors are constitutively expressed in the nucleus and this type of receptor would not be amenable to a nuclear translocation assay. The activities of nuclear receptors may be dependent upon complex interactions with a number of coregulatory proteins, commonly known as coactivators or corepressors, and modifications by post-translational means. Cell type-specific expression levels of receptors and coregulators may contribute to some, but not all, of the molecular bases for gene and functional selectivity of receptor activity. Therefore selecting a cell line that expresses both the target receptor and the necessary cofactors may be required to design an appropriate assay. 

The development of this type of nuclear translocation assay can be relatively simple because the manufacturers have identified conditions to maximize the response. While a cell titration coupled with agonist and/or antagonist titrations is still required, the optimal conditions for the chemistry involved in the detection are generally provided by the assay reagents. In addition, time and temperature of incubations should be optimized for each receptor and cell line. A sample protocol of a 1536-well plate assay in which the detection of the nuclear translocation of GR is measured is listed below ... 

Examples best illnstrate the power of HCS. There is a tremendous variety of assays possible. This includes chemotaxis, morphological changes, nuclear translocation, snbcellnlar localization, cell-to-cell communication, cell viability, toxicity, micronuclei formation, cell cycle arrest, and receptor internalization. An NF-kB nuclear translocation assay, an apoptosis assay, and a gap jnnction screen are described below. Only the gap jnnction assay was performed at sanofi-aventis the other two examples were taken from an author s experience at Prelnx with the development of the precnrsor to the INCell 3000. 

Example 1 NF-kB Nuclear Translocation Assay (from Prelux)... 

The translocation of a nuclear hormone receptor to the nucleus is an early stage of receptor activation. A number of assay technologies can detect this nuclear translocation event. For example, using... 

Many inflammatory cytokines including IL-8 are regulated at transcriptional levels, and a variety of transcription factors such as nuclear factor-K B (NFkB) and activator protein-1 (AP-1) play important roles in such processes. Abe and coworkers [71] demonstrated that CAM repressed TNF-a-induced AP-1 activation in human bronchial epithelial cells. We studied the effect of EM and CAM on the phorbol myristate acetate (PMA)-induced activation of NFkB and AP-1. Pretreatment of EM and CAM before the PMA treatment showed an inhibitory effect on both of the transcription factors as assessed by electrophoretic mobility shift assay (EMSA) (Fig. 14) [20]. In contrast, the macrolides showed no effect on the activation of cyclic AMP-responsive element binding protein (CREB), suggesting that the suppressive effect on some transcription factors is specific. We further evaluated the effect of EM on the phosphorylation of inhibitor of NFkB (IkB), which is a crucial step for transactivation of NFkB. EM did not influence the phosphorylation processes in vitro (Okazaki et at, unpublished data, January 2001). These data suggest that EM acts at the process of nuclear translocation of... 

Although PB treatment induces the nuclear translocation and transcriptional activity of CAR, results from ligand-binding assays have indicated that neither PB nor known PB metabolites are... 

To investigate the effects of drugs on NFkB activation at the molecular level, the Electric Mobility Shift Assay (EMSA) is a useful read-out system. With this technique the nuclear localization of this transcription factor following activation and subsequent translocation can... 

In some experiments concerned with the mechanism by which tryptophan acted to improve hepatic protein synthesis after toxic injury, the ability of tryptophan to stimulate hepatic mRNA synthesis, nucleocytoplasmic translocation of RNA in vitro, and nuclear envelope nucleoside triphosphatase activity after hepatotoxic injury was measured.188 Nucleoside triphosphatase (Mg2+-dependent adenosine triphosphatase, EC 3.6.1.3.1) was assayed since it is present in mammalian liver nuclear envelopes,224 and there is evidence that this enzyme is involved in nucleocytoplasmic translocation of RNA.221 All of these parameters were elevated significantly by tryptophan after agents such as actinomycin D, cordycepin, ethionine, puromycin, and hypertonic NaCl demonstrated a curative effect by tryptophan, but not after tryptophan following CC14, NaF, and sparsomycin demonstrated no improvement with tryptophan. These findings emphasized the importance of the role that tryptophan plays in stimulating the availability of cytoplasmic... 

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