Phospho-p44/42 MAPK (Erk1/2) (

9101:

Flow , Immunofluorescence* , Immunoprecipitation , Western Blotting

* Product-specific protocol.

Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody detects endogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when phosphorylated either individually or dually at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2). The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP Kinase, and does not cross-react with non-phosphorylated Erk1/2.

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Thr202/Tyr204 of human p44 MAP kinase.

Western Blotting

Specificity and sensitivity of Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody. The antibody reacts specifically with as little as 0.25 ng of phosphorylated p42 MAP kinase and does not cross-react with up to 4 µg of nonphosphorylated p42 MAP kinase.

Western Blotting

Western blot analysis of whole-cell extracts from unstarved wild-type mouse embryonic fibroblasts (MEFs) treated with the indicated combinations of basic Fibroblast Growth Factor (bFGF #9952, 100 ng/ml for 30 minutes), Platelet-Derived Growth Factor (PDGF #9909, 100 ng/ml for 30 minutes), MEK1 Inhibitor (PD98059 #9900, 50 µM, 2 hour pre-treatment), and MEK1/2 Inhibitor (U0126 #9903, 10 µM, 2 hour pre-treatment), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody #9101 (upper panel) and p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower panel).

Flow Cytometry

Flow cytometric analysis of Jurkat cells, untreated (green) or PMA-treated (blue), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody compared to a nonspecific negative control antibody (red).

Flow Cytometry

Phosphorylated MEK and Erk were assayed in human peripheral blood lymphocytes stimulated with PMA in the presence or absence of the Raf inhibitor BAY 37-9751 or the MEK inhibitor U0126 #9903. BAY 37-951 blocked PMA-stimulated phosphorylation of both MEK and Erk, consistent with inhibition at the level of Raf, while U0126 blocked phosphorylation of Erk only, consistent with inhibition at the level of MEK. From Chow, S. et al. (2001) Cytometry 46, 72-78.

IF-IC

Confocal immunofluorescent analysis of NIH/3T3 cells either U0126-treated (left) or PDGF-treated (right) and labeled with Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red).

Mitogen-activated protein kinases (MAPKs) are a widely conserved family of serine/threonine protein kinases involved in many cellular programs such as cell proliferation, differentiation, motility, and death. The p44/42 MAPK (Erk1/2) signaling pathway can be activated in response to a diverse range of extracellular stimuli including mitogens, growth factors, and cytokines (1-3) and is an important target in the diagnosis and treatment of cancer (4). Upon stimulation, a sequential three-part protein kinase cascade is initiated, consisting of a MAP kinase kinase kinase (MAPKKK or MAP3K), a MAP kinase kinase (MAPKK or MAP2K), and a MAP kinase (MAPK). Multiple p44/42 MAP3Ks have been identified, including members of the Raf family, as well as Mos and Tpl2/Cot. MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate p44 and p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of p44/42 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). p44/42 are negatively regulated by a family of dual-specificity (Thr/Tyr) MAPK phosphatases, known as DUSPs or MKPs (10), along with MEK inhibitors such as U0126 and PD98059.

1. Roux, P.P. and Blenis, J. (2004) Microbiol Mol Biol Rev 68, 320-44.
2. Baccarini, M. (2005) FEBS Lett 579, 3271-7.
3. Meloche, S. and Pouysségur, J. (2007) Oncogene 26, 3227-39.
4. Roberts, P.J. and Der, C.J. (2007) Oncogene 26, 3291-310.
5. Rubinfeld, H. and Seger, R. (2005) Mol Biotechnol 31, 151-74.
6. Murphy, L.O. and Blenis, J. (2006) Trends Biochem Sci 31, 268-75.
7. Dalby, K.N. et al. (1998) J Biol Chem 273, 1496-505.
8. Marais, R. et al. (1993) Cell 73, 381-93.
9. Kortenjann, M. et al. (1994) Mol Cell Biol 14, 4815-24.
10. Owens, D.M. and Keyse, S.M. (2007) Oncogene 26, 3203-13.

• Schulze, A. et al. (2001) Genes. Dev. 15, 981-94. Applications: Western Blotting
• Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4. Applications: Western Blotting
• Zhang, Y.W. et al. (2003) Proc. Natl Acad. Sci. USA 100, 12718-23. Applications: Western Blotting
• Patrucco, E. et al. (2004) Cell 118, 375-87. Applications: Western Blotting
• Yu, C. et al. (2002) Cancer Res. 62, 188-99. Applications: Western Blotting
• Dai, Y. et al. (2001) Cancer Res. 61, 5106-15. Applications: Western Blotting
• Chow, S. et al. (2001) Cytometry 46, 72-8. Applications: Flow Cytometry
• Kawagoe, T. et al. (2008) Nat Immunol 9, 684-91. Applications: Western Blotting
• Abe, T. et al. (2007) J Virol 81, 8953-66. Applications: Western Blotting
• Tsuda, M. et al. (2008) Glia 56, 378-86. Applications: IF-IC (In Cells)
• Moritz, A. et al. (2010) Sci Signal 3, ra64. Applications: Western Blotting

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• 9194 p44/42 MAPK (Erk1/2) Control Cell Extracts
• 4370 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP^® Rabbit mAb
• 4376 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb
• 4377 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (197G2) Rabbit mAb
• 9106 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb
• 4695 p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb
• 2321 Phospho-(Thr) MAPK/CDK Substrate Mouse mAb
• 2325 Phospho-MAPK/CDK Substrates (PXS*P or S*PXR/K) (34B2) Rabbit mAb
• 7074 Anti-rabbit IgG, HRP-linked Antibody
• 7720 Prestained Protein Marker, Broad Range (Premixed Format)
• 7727 Biotinylated Protein Ladder Detection Pack
• 7003 20X LumiGLO^® Reagent and 20X Peroxide
• 1150 Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Blocking Peptide

For Research Use Only. Not For Use In Diagnostic Procedures.