The Pathscan® Multiplex Western Cocktail III Detection Kit offers a unique method to assay the activation of multiple pathways on one membrane without stripping and reprobing. This method saves the user valuable time, while increasing accuracy and minimizing reagent waste. The system allows the user to simultaneously detect levels of phospho- Stat1, phospho-SAPK/JNK, phospho-S6 ribosomal protein and phospho-HSP27. The kit also includes Pin1 antibody to control protein loading. In addition, each Pathscan® Multiplex Western Cocktail Detection Kit contains treated and untreated cell lysates and the Phototope®-HRP Western Detection System. The kit includes enough primary and secondary antibodies to perform five Western blot experiments.
Each phospho-antibody in this kit recognizes only the phosphorylated form of its specific target. The Pin1 antibody detects total levels of target protein to control for protein loading. All the antibodies in this kit detect endogenous levels of target proteins.
Antibodies are produced by immunizing animals with synthetic peptides. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.
Stat1, while activated in response to a large number of ligands, appears to be essential for responsiveness to IFN-alpha and IFN-gamma (1-3).
Phosphorylation of Stat1 at Tyr701 induces Stat1 dimerization, nuclear translocation and DNA binding (4). Stat1 has been found to be inappropriately activated in many tumors (5).
The stress-activated protein kinase/Jun-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 (6,7). SAPK/JNK, when active as a dimer, can translocate to the nucleus where it regulates transcription through its effects on c-Jun, ATF-2 and other transcription factors (8).
To effectively promote growth and cell division in a sustained manner, growth factors and mitogens must upregulate translation (9,10). Growth factors and mitogens induce the activation of p70 S6 kinase, which in turn phosphorylate the S6 ribosomal protein. Phosphorylation of S6 correlates with an increase in translation, particularly of mRNAs with an oligopyrimidine tract in their 5' untranslated regions (10). This group of mRNAs (5'TOP) encodes proteins involved in cell cycle progression and proteins that are part of the translational machinery, such as ribosomal proteins and elongation factors (10,11).
Heat shock protein (HSP) 27 is one of the small HSPs, regulated at both the transcriptional and posttranslational levels (12). In response to stress, the expression level of HSP27 increases several-fold to confer cellular resistance to the adverse environmental change. HSP27 is also phosphorylated at serines 15, 78 and 82 by MAPKAP kinase 2 as a result of p38 MAP kinase pathway activation (13,14).
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