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Analyses of MAPKAPK2 (pT334) expression by Human and Mouse Cells. Human Cells Panel 1a: Flow Cytometric analysis of MAPKAPK2 (pT334) in peripheral blood lymphocytes (PBL). Whole blood cells were not stimulated (dashed line histogram) or stimulated (solid line histogram) with 400 nM Phorbol 12-Myristate 13-Acetate (PMA; Sigma, Cat. No. P8139; 15 min, 37°C). Cells were fixed in 1X BD Phosflow™ Lyse/Fix Buffer (Cat. No. 558049; 10 min, 37˚C) and permeabilized in BD Phosflow™ Perm Buffer III (Cat. No. 558050) on ice (30 min). Cells were stained with BD Phosflow™ Alexa Fluor® 647 Mouse Anti-MAPKAPK2 (pT334) (Cat. No. 562472) Ab. Fluorescence histograms showing MAPKAPK2 (pT334) expression were generated for gated events with the light scatter characteristics of intact lymphocytes using a BD FACSCanto™ II Flow Cytometer. Panel 1b: Western blot analysis of MAPKAPK2 (pT334) expressed by peripheral blood mononuclear cells (PBMC). Lysates from 1X10^6 untreated (C) and PMA-treated (T) PBMC were blotted using Purified Mouse Anti-MAPKAPK2 (pT334) Ab (2 µg/ml, Cat. No. 562469), HRP Goat Anti-Mouse Ig (Cat. No. 554002) and a chemiluminescent detection system. MAPKAPK2 (pT334) were identified as ~49 kDa bands by Western blotting. Mouse Cells Panel 2: Splenocytes were not stimulated (dashed line histogram) or stimulated (solid line histogram) with PMA (50 nM, 15 min, 37°C). Cells were fixed, permeabilized, stained with Alexa Fluor® 647 Mouse anti-MAPKAPK2 (pT334) Ab and analyzed by flow cytometry as described above.
BD™ Phosflow Alexa Fluor® 647 Mouse anti-MAPKAPK-2 (pT334)
BD™ Phosflow Alexa Fluor® 647 Mouse anti-MAPKAPK-2 (pT334)
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Preparation And Storage
Product Notices
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
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- The Alexa Fluor®, Pacific Blue™, and Cascade Blue® dye antibody conjugates in this product are sold under license from Molecular Probes, Inc. for research use only, excluding use in combination with microarrays, or as analyte specific reagents. The Alexa Fluor® dyes (except for Alexa Fluor® 430), Pacific Blue™ dye, and Cascade Blue® dye are covered by pending and issued patents.
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Companion Products
The P24-694 monoclonal antibody specifically binds to the phosphorylated T334 site (pT334) of MAPKAPK-2. MAPKAPK-2 is a serine/threonine protein kinase. This ~49 kDa member of the MAPKAPK family of protein kinases is also known as mitogen-activated protein kinase-activated protein kinase 2. MAPKAPK-2 is phosphorylated and activated by p38 MAP kinase in response to stress, cytokines and chemokines. MAPKAPK-2 is phosphorylated on multiple sites including Thr222, Ser272 and Thr334. Phosphorylation of any two of these three amino acid residues seems to be required for the activation of this kinase that serves multiple cellular functions. Phosphorylation of Thr334 was reported to be essential for nuclear export of the heterodimer formed between p38 MAPK and MAPKAPK-2. Mice deficient in MAPKAPK-2 have been shown to be protected from ischemic injury. MAPKAPK-2 is also reported to serve as a cell cycle checkpoint kinase in response to UV irradiation. The heat shock protein, HSP27 was shown to be one of the major substrates of MAPK and MAPKAPK-2.
Development References (8)
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Clifton AD, Young PR, Cohen P. A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Lett. 1996; 392(3):209-214. (Biology). View Reference
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Engel K, Kotlyarov A, Gaestel M. Leptomycin B-sensitive nuclear export ofMAPKAP kinase 2 is regulated by phosphorylation. EMBO J. 1998; 17(12):3363. (Biology). View Reference
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Heidenreich O, Neininger A, Schratt G, et al. MAPKAP kinase 2 phosphorylates serum response factor in vitro and in vivo. J Biol Chem. 1999; 274(20):14434-14443. (Biology). View Reference
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Krump E, Sanghera JS, Pelech SL, Furuya W, Grinstein S. Chemotactic peptideN-formyl-met-leu-phe activation of p38 mitogen-activated protein kinase (MAPK)and MAPK-activated protein kinase-2 in human neutrophils. J Biol Chem. 1997; 272(2):937. (Biology). View Reference
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Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE, Yaffe MB. MAPKAP kinase-2 is acell cycle checkpoint kinase that regulates the G2/M transition and S phaseprogression in response to UV irradiation. Mol Cell. 2005; 17(1):37. (Biology). View Reference
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Meng W, Swenson LL, Fitzgibbon MJ, et al. Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export. J Biol Chem. 2002; 277(40):37401-37405. (Biology). View Reference
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Rouse J, Cohen P, Trigon S, et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell. 1994; 78(6):1027-1037. (Biology). View Reference
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Wang X, Xu L, Wang H, Young PR, Gaestel M, Feuerstein GZ.. Mitogen-activatedprotein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain fromischemic injury in mice. J Biol Chem. 2002; 277(46):43968. (Biology). View Reference
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For Research Use Only. Not for use in diagnostic or therapeutic procedures.