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Western blot analysis of CREB (pS133) / ATF-1 (pS63). Lysates from control (left panel) and PMA-activated (right panel) Jurkat cells (Human T-cell leukemia; ATCC TIB-152) were probed with the purified mouse anti-CREB (pS133) / ATF-1 (pS63) antibody at concentrations of 0.25 (lanes 1 and 4), 0.125 (lanes 2 and 5), and 0.06 µg/ml (lanes 3 and 6). CREB (pS133) is identified as a band of 43 kDa in the PMA-treated cells. Another band migrating below it has been identified as ATF-1 (pS63). Treatment with lambda phosphatase removes all bands in blots of control and PMA-activated lysates (data not shown), demonstrating the antibody's specificity for the indicated phosphorylated proteins.
BD Pharmingen™ Purified Mouse Anti-CREB (pS133) / ATF-1 (pS63)
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Western blot: Please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml
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- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
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Transcription of various genes is regulated by the cyclic AMP (cAMP) signal transduction pathway through a family of cAMP Responsive Element (CRE)-Binding transcription factors that include CREB, CREM (CRE Modulator), and ATF-1 (Activating Transcription Factor 1). The genes for these transcription factors encode multiple isoforms that are created by alternative splicing, alternative initiation codons, and alternative intronic promoters. Homo- and heterodimers of these members of the basic domain-and-leucine zipper superfamily of proteins bind the palindromic TGACGTCA sequence of CRE. CREB is expressed in all somatic cells, and many different stimuli activate CREB by phosphorylation of its serine 133 (S133). The transcriptional activity and specificity of CREB are regulated by the signaling pathways initiated by the various stimuli. Transcriptional regulation by CREB family members has been implicated in many physiological responses to extracellular and environmental stimuli, such as memory, tissue growth and development, and homeostasis.
The J151-21 monoclonal antibody recognizes the phosphorylated S133 of human CREB in addition to cross-reacting with S63 of human ATF-1.
Development References (1)
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Shaywitz AJ, Greenberg ME. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem. 1999; 68:821-861. (Biology).
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