Cellomics HCS Reagent Kit for multiplex detection of phosphorylated c-Jun and phosphorylated Jun N-terminal kinase (JNK).

Nuclei	Phospho-c-Jun	Phospho-JNK
Measure three parameters in the same cells			Determine dose responses

Description

The Cellomics Phospho-c-jun and Phospho-JNK Detection Kit enables quantitation of phosphorylated c-jun (phospho-c-jun) and phosphorylated Jun N-terminal kinase (phospho-JNK) in the nucleus. Phospho-JNK, also referred to as stress-activated protein kinase 1 (SAPK), and phospho-c-jun are measured directly using a fixed end-point assay based on immunofluorescence detection in cells grown on standard high-density microplates. This kit contains an anti-phospho-c-jun primary antibody (mouse monoclonal), anti-phospho-JNK primary antibody (rabbit monoclonal), DyLight Fluor-conjugated Secondary Antibodies and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.

Mitogen-activated protein kinase (MAPK) pathways consist of four major groupings and numerous related proteins. The four major groupings are JNK, Erk, p38 and ERK5 cascades. Signals from cell surface receptors are transduced directly or via small G-proteins, such as Ras and Rac, to multiple tiers of protein kinases that amplify these signals and regulate each other. These MAPK cascades eventually result in activation of numerous transcription factors that regulate genes involved in apoptosis, inflammation and cell growth and differentiation. JNK activation is mediated by tumor-necrosis factor alpha (TNFa), UV or protein synthesis inhibitors, resulting in phosphorylation of JNK at Thr183/Thr185 residues.

The transcription factor c-Jun is activated through phosphorylation in the activation domain by Jun-N-terminal kinases (JNKs). Phosphorylated Jun family members then form homodimers or heterodimeric complexes with Fos, composing the AP-1 transcription factor, which migrates into the nucleus. The AP-1 transcription factor has been implicated in stress induced apoptosis, heat shock, T-cell activation, cellular responses to inflammatory cytokines, cellular transformation and ischemia/reperfusion. c-Jun is activated by a wide variety of stimuli, including anisomycin, epidermal growth factor, hydrogen peroxide and TNFa.

This kit was optimized using the Thermo Scientific ArrayScan HCS Reader and the Compartmental Analysis BioApplication Software Module but can be used with other Cellomics BioApplications. Thus, automated plate-handling, focusing, cell image acquisition and data analysis are combined in one HCS system to assay for compounds that regulate phospho-c-jun and phospho-JNK expression. In addition to HCS instruments, cells stained using these kit reagents can be viewed and analyzed by fluorescence and confocal microscopes.

Figure 1. Staining of phospho-c-jun and phospho-JNK in NIH 3T3 cells treated with media (non-treated) or with anisomycin (1 µM) for 30 minutes. Cells were stained according to the kit protocol and imaged using the ArrayScan VTI HCS Reader.

Figure 2. Dose response curves for phospho-c-jun and phospho-JNK in NIH 3T3 cells. The average intensities of phospho-c-jun and phospho-JNK in the nucleus in response to various concentrations of indicated drugs were plotted and their EC50 calculated.

Kit Contents

Ordering Information

Screening size kits and components (bulk and custom) available with special pricing. Please call.

To order Cellomics HCS Reagent Kits, please call 800-874-3723 in the U.S.
or contact your local distributor of Thermo Scientific Products.

Choose "Order Online" in table to see pricing and order online (U.S. only).

See also:
Phospho-c-Jun Kit
NFkB and Phospho-c-Jun Multiplex Kit

See all Cellomics HCS Reagent Kits