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GFP-MAPKAP-k2
The GFP-MAPKAP-k2 assay provides a live-cell screening resource to measure the response of the p38 MAP kinase (p38 MAPK) cell signaling pathway. The intracellular translocation of a GFP-MAPKAP-k2 fusion protein is monitored in the presence of an agonist. The assay consists of an extensively validated stably transfected cell line and expression vector. It can be used with automated and traditional confocal and epifluorescent microscopes.
.Validated stable cell line: start screening immediately without having to spend months establishing a cell line in-house.
Expression vector: offers the flexibility to work with transients and alternative host cell lines
Complete right to use: no additional license negotiations are required prior to using the assay
Direct observation of MAPKAP-k2 movement and location: permits pathway dissection in correct physiological context, identifies inhibitors of p38 MAPK signaling pathway
Cellular assay: identifies potentially toxic compounds without the need for additional reagents
Utilizes Aequorea victorea GFP: the established benchmark fluorescent protein technology
The p38 MAPK signaling pathway mediates a variety of stress and inflammatory responses in eukaryotic cells and is activated by factors including heat shock, UV light, pro-inflammatory cytokines, and protein synthesis inhibitors such as anisomycin.
Upon p38 MAPK-mediated phosphorylation, MAPKAP-k2 translocates from the nucleus to the cytoplasm and can be used to report on the upstream p38 MAPK signaling pathway.
In the cytoplasm, MAPKAP-k2 stimulates the production of pro-inflammatory cytokines, including TNFa and IL-6, by affecting mRNA translation and/or stability. Therefore, translocation of MAPKAP-k2 from the nucleus to the cytoplasm is a good measure of the inflammatory output from the p38 MAPK cascade.
The degree of pathway response when challenged with test compounds is quantitated by measuring the cytoplasmic-to-nuclear intensity ratio of the GFP-MAPKAP-k2 fusion protein.
GFP-MAPKAP-k2 assay was designed as a live-cell system using image acquisition and analysis on the IN Cell Analyzer 3000 and
IN Cell Analyzer 1000. However, this assay can also be performed in a fixed-cell format on these platforms. Alternatively, the assay may be used in conjunction with other automated subcellular imaging platforms or fluorescence microscopes. |
| Fig 2. Agonist dose response curve. The reference agonist employed in the assay is anisomycin, EC50 = 47 nM (determined on IN Cell Analyzer 3000 using the Nuclear Trafficking Analysis Module). Error bars indicate SD, n = 8. The assay has also been configured in antagonist format.
For more information please consult the catalog entry. |
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