Figure 2. c-Jun fusion protein was used as substrate to measure JNK1 kinases activity in a radiometric assay using the following reaction conditions: 25 mM Tris-HCl (pH7.5), 10 mM MgCl2, 5 mM β-glycerophosphate, 0.1 mM Na3VO4, 2 mM DTT, 50 μM ATP, Substrate: c-Jun fusion protein 400 ng/μL, and variable amounts of JNK1 kinases.
c-Jun Fusion Protein serves as a useful substrate for SAPK/JNK, which will phosphorylate it at Ser63 and Ser73 (1). It is expressed as a recombinant protein fusion to amino acid residues corresponding to c-Jun codons 1-89.
Apparent Molecular Weight: 37 kDa
c-Jun Fusion Protein at a concentration of 0.5 µg/µl in a 20 µl reaction can be phosphorylated using active SAPK in an in vitro kinase assay with 1X Kinase Buffer (#9802) and 200 µM ATP (#9804). After a 30-minute assay at 30ºC, phosphorylation can be detected by Western blot with phospho-specific c-Jun antibodies (#9164 and #9261).
Store at -20°C.
The purified protein was identified by SDS-PAGE and Western blot analysis, using c-Jun Antibody #9162, to be greater than 95% c-Jun Fusion Protein.
Isolated from a strain of E. coli that carries the coding sequence for human c-Jun residues 1-89 (kindly provided by Dr. J.R. Woodgett). Protein was purified by affinity chromatography.
c-Jun is a member of the Jun family containing c-Jun, JunB, and JunD, and is a component of the transcription factor activator protein-1 (AP-1). AP-1 is composed of dimers of Fos, Jun, and ATF family members and binds to and activates transcription at TRE/AP-1 elements (reviewed in 1). Extracellular signals including growth factors, chemokines, and stress activate AP-1-dependent transcription. The transcriptional activity of c-Jun is regulated by phosphorylation at Ser63 and Ser73 through SAPK/JNK (reviewed in 2). Knock-out studies in mice have shown that c-Jun is essential for embryogenesis (3), and subsequent studies have demonstrated roles for c-Jun in various tissues and developmental processes including axon regeneration (4), liver regeneration (5), and T cell development (6). AP-1 regulated genes exert diverse biological functions including cell proliferation, differentiation, and apoptosis, as well as transformation, invasion and metastasis, depending on cell type and context (7-9). Other target genes regulate survival, as well as hypoxia and angiogenesis (8,10). Research studies have implicated c-Jun as a promising therapeutic target for cancer, vascular remodeling, acute inflammation, and rheumatoid arthritis (11,12).
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