Product Pathways - MAPK Signaling
SAPK/JNK Kinase Assay Kit (Nonradioactive) #9810
| Kit Includes | Quantity | Source |
|---|---|---|
| c-Jun Fusion Protein Beads # 9811 | 800 microliters | |
| Kinase Buffer (10X) # 9802 | 15 milliliters | |
| Cell Lysis Buffer (10X) # 9803 | 15 milliliters | |
| ATP (10 mM) # 9804 | 50 microliters | |
| Anti-rabbit IgG, HRP-linked Antibody # 7074 | 50 microliters | Goat |
| Anti-biotin, HRP-linked Antibody # 7075 | 100 microliters | Goat |
| Biotinylated Protein Ladder Detection Pack # 7727 | 100 microliters | |
| 20X LumiGLO® Reagent and 20X Peroxide # 7003 | 5 milliliters | |
| Phospho-c-Jun (Ser63) Antibody (unique to kit) | 100 microliters |
Molecular Weight
35, 37 kDa
Reactivity
H M R
Reactivity Key: H=Human M=Mouse R=Rat
Description
Nonradioactive SAPK/JNK Assay Kit provides all the reagents necessary to measure SAPK/JNK activity in the cell. A c-Jun fusion protein linked to agarose beads is used to pull down SAPK enzyme from cell extracts. Upon addition of kinase buffer and ATP, SAPK phosphorylates the c-Jun substrate. Phospho-c- Jun (Ser63) Antibody can then be used to measure SAPK activity by immunoblotting.
Western Blotting
SAPK/JNK activity of UV, arsenite and anisomycin-treated SK-N-MC cell extracts was analyzed by IP/Kinase assay. Phosphorylation of c-Jun at Ser63 was visualized by immunoblotting with Phospho-c-Jun (Ser63) Antibody
Western Blotting
SAPK-induced phosphorylation of c-Jun was measured by quantitative immunoblotting with Phospho-c-Jun (Ser63) Antibody (A) and compared to direct measurement of phosphate incorporation using [gama-32P]-ATP (B). (Note exposure time difference)
Background
The stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) is potently and preferentially activated by a variety of environmental stresses, including UV and gamma radiation, ceramides, inflammatory cytokines and in some instances, by growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4-7, which then phosphorylate and activate the SAPK/JNK kinase (2). Stress signals are delivered to this cascade by small GTPases of the Rho family (Rac, Rho, cdc42) (3). Both Rac1 and cdc42 mediate the stimulation of MEKKs and MLKs (3). Alternatively, MKK4-7 can be activated by a pathway independent of small GTPases via stimulation of a member of the germinal center kinase (GCK) family (4). There are three SAPK/JNK genes with further diversification resulting from alternative splicing (3). Active SAPK/JNK dimers can translocate to the nucleus to regulate transcription through its effects on c-Jun, ATF-2 and other transcription factors (3,5).
- Davis, R.J. (1999) Biochem. Soc. Symp. 64, 1-12.
- Ichijo, H. (1999) Oncogene 18, 6087-6093.
- Kyriakis, J.M. and Avruch, J. (2001) Physiol. Rev. 81, 807-869.
- Kyriakis, J.M. (1999) J. Biol. Chem. 274, 5259-5262.
- Leppa, S. and Bohmann, D. (1999) Oncogene 18, 6158-6162.
- Whitmarsh, A.J. and Davis, R.J. (1998) Trends Biochem. Sci. 23, 481-485.
Application References
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