SAPK-induced phosphorylation of c-Jun was measured by quantitative immunoblotting using Phospho-c-Jun (Ser63) Antibody (upper) and c-Jun (L70B11) Mouse mAb #2315 (lower). Lysate titrations were performed by immuoprecipitating phospho-SAPK/JNK from the indicated amounts of HeLa cell extracts, untreated or UV-treated. Phosphorylation of c-Jun at Ser63 is observed in UV-treated lysates.
The SAPK/JNK Kinase Assay Kit (Nonradioactive) provides all the reagents necessary to measure SAPK/JNK activity in the cell. A phospho-SAPK/JNK Rabbit mAb linked to agarose beads is used to pull down SAPK enzyme from cell extracts. Upon addition of kinase buffer, c-Jun fusion protein and ATP, SAPK phosphorylates the c-Jun substrate. Phospho-c-Jun (Ser63) Antibody can then be used to measure SAPK activity by immunoblotting.
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, growth factors and GPCR agonists (1-6). As with the other MAPKs, the core signaling unit is composed of a MAPKKK, typically MEKK1-MEKK4, or by one of the mixed lineage kinases (MLKs), which phosphorylate and activate MKK4/7. Upon activation, MKKs 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 in a GTPase-independent mechanism via stimulation of a germinal center kinase (GCK) family member (4). There are three SAPK/JNK genes each of which undergoes alternative splicing, resulting in numerous isoforms (3). SAPK/JNK, when active as a dimer, can translocate to the nucleus and regulate transcription through its effects on c-Jun, ATF-2, and other transcription factors (3,5).
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