Product Pathways - MAPK Signaling

Phospho-Erk1/2 Pathway Sampler Kit #9911

• pathway
• more info
• application references
• datasheet PDF
• MSDS PDF
• protocols

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IHC-P=Immunohistochemistry (Paraffin)  IF-IC=Immunofluorescence (Immunocytochemistry)  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Mi=Mink  Hm=Hamster  B=Bovine  Dr=Drosophila  Z=Zebra Fish  Dg=Dog

Specificity / Sensitivity

Each antibody in the Phospho-Erk1/2 Pathway Sampler Kit recognizes only the phosphorylated form of its specific target.

Western Blotting

Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA as indicated, using Phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370 (upper), or p44/42 MAP Kinase (137F5) Rabbit mAb #4695 (lower).

Western Blotting

Western blot analysis of extracts from TPA-treated (200 nM) 293 cells using Phospho-p90RSK (Ser380) Antibody #9341 (upper) or control RSK1/RSK2/RSK3 (32D7) Rabbit mAb #9355 (lower).

Western Blotting

Western blot analysis of extracts from NIH/3T3, HeLa and COS cells, untreated or treated with TPA, using Phospho-c-Raf (Ser338) (56A6) Rabbit mAb #9427.

Western Blotting

Western blot analysis of extracts from untreated or TPA-treated HeLa and NIH/3T3 cells, using Phospho-MEK1/2 (Ser217/221) (41G9) Rabbit mAb #9154 (upper), or MEK1/2 Antibody #9122 (lower).

Western Blotting

Western blot analysis of Elk-1 fusion protein expressed from E. coli with or without phosphorylation by purified Erk2 enzyme using Phospho-Elk-1 (Ser383) Antibody (upper) #9181 or control Elk-1 Antibody #9182 (lower).

IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370.

Flow Cytometry

Flow cytometric analysis of Jurkat cells, U0126-treated (blue) or PMA-treated (green), using Phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370.

IF-IC

Confocal immunofluorescent analysis of C2C12 cells, U0126-treated (upper, #9903 10 μM for 1 h) or TPA-treated (lower, #9905 200 nM for 15 min), using Phospho-p44/42 MAPK (Thr202/Tyr204) (D13.14.4E) Rabbit mAb #4370 (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin (red). Blue pseudocolor = DRAQ5™ (fluorescent DNA dye).

Source / Purification

Polyclonal and monoclonal antibodies are produced by immunizing rabbits with synthetic phospho-peptides corresponding to residues surrounding Ser338 of human Raf, Ser217/221 of human MEK1/2, Thr202/Tyr204 of human p44 MAP kinase, Ser380 of human p90RSK or Ser383 of human Elk-1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

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), a MAP kinase kinase (MAPKK), and a MAP kinase. While multiple ERK1/2 MAP3Ks have been identified, including the Raf family, Mos, and Tpl2/Cot, MEK1 and MEK2 are the primary MAPKKs in this pathway (5,6). MEK1 and MEK2 activate ERK1/p44 and ERK2/p42 through phosphorylation of activation loop residues Thr202/Tyr204 and Thr185/Tyr187, respectively. Several downstream targets of ERK1/2 have been identified, including p90RSK (7) and the transcription factor Elk-1 (8,9). ERK1/2 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 Pouyssegur, 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.

Application References

Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!

Companion Products

• 9422 c-Raf Antibody
• 9126 MEK1/2 (47E6) Rabbit mAb
• 4695 p44/42 MAP Kinase (137F5) Rabbit mAb
• 9355 RSK1/RSK2/RSK3 (32D7) Rabbit mAb
• 9182 Elk-1 Antibody
• 9160 MEK1/2 HeLa Control Cell Extracts
• 7071 Phototope^®-HRP Western Blot Detection System, Anti-rabbit IgG, HRP-linked Antibody
• 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

Rabbit Monoclonals Produced UsingTechnology from Epitomics, Inc. UnderU.S. patent No. 5,675,063.