Phospho-JNK Detection Kit

Phospho-JNK, also referred to as stress-activated protein kinase 1 (SAPK), is 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-JNK primary antibody (rabbit monoclonal), a goat anti-rabbit secondary antibody conjugated to DyLight™ 549 Fluorophore and various other reagents and buffers required for immunofluorescence staining for high-content screening (HCS) assays.



Figure 2. Staining of phospho-JNK in HeLa and HepG2 cells treated with media (non-treated) or with anisomycin (2.25 µM) for 30 minutes. Cells were stained according to the kit protocol and imaged using the ArrayScan VTI HCS Reader.

Mitogen-activated protein kinase (MAPK) pathways consist of four major groupings and numerous related proteins, which constitute interrelated signal transduction cascades activated by stimuli such as growth factors, stress, cytokines and inflammation. 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, cell growth and differentiation. JNK activation is mediated by tumor-necrosis factor alpha (TNF-a), UV or protein synthesis inhibitors such as anisomycin, resulting in phosphorylation of JNK at Thr183/Thr185 residues.

This kit was optimized using the Cellomics 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-JNK expression. In addition to HCS instruments, cells stained using reagents in this kit can be viewed and analyzed by fluorescence and confocal microscopes.

Figure 3. Dose response curves for phospho-JNK in HepG2 and HeLa cells. The difference in average intensities of phospho-JNK in the nucleus and cytoplasm is plotted in response to anisomycin concentration in cells treated for 30 minutes. EC₅₀ for HepG2 = 120.3 nM. EC₅₀ for HeLa = 33.5 nM.

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