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9911
Phospho-Erk1/2 Pathway Sampler Kit

Phospho-Erk1/2 Pathway Sampler Kit #9911

Western Blotting Image 1

Western blot analysis of extracts from NIH3T3, HeLa and COS cells, untreated or treated with TPA, using Phospho-c-Raf (Ser338) (56A6) Rabbit mAb.

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Western Blotting Image 2

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

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Western Blotting Image 3

Western blot analysis of extracts from COS cells, untreated or treated with either U0126 #9903 (10 µM for 1h) or TPA #4174 (200 nM for 10 m), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 and p44/42 MAPK (Erk1/2) (3A7) Mouse mAb #9107.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines, starved overnight and either untreated (-) or treated (+) with TPA #4174 (200 nM, 15 min), Human Epidermal Growth Factor (hEGF) #8916 (100 ng/mL, 15 min), or Human Platelet-Derived Growth Factor BB (hPDGF-BB) #8912 (100 ng/mL, 15 min) as indicated, using Phospho-p90RSK (Ser380) (D3H11) Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from 293 cells, transfected with wt MSK1 or mutant MSK1, untreated or TPA-treated (200 nM), using Phospho-MSK1 (Thr581) Antibody.

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Western Blotting Image 6

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

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Western Blotting Image 7

Western blot analysis of extracts from 293, NIH/3T3 and C6 cells, treated with λ phosphatase or TPA #4174 as indicated, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (upper), or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (lower).

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IHC-P (paraffin) Image 8

Immunohistochemical analysis of paraffin-embedded human colon carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-p90RSK (Ser380) (D3H11) Rabbit mAb.

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Western Blotting Image 9

Western blot analysis of extracts from 293 cells, untreated, UV-treated or calyculin A-treated, using Phospho-MSK1 (Thr581) Antibody.

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IHC-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 11

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Phospho-p90RSK (Ser380) (D3H11) Rabbit mAb.

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Western Blotting Image 12

Western blot analysis of extracts from 293 cells untreated or treated with UV and HeLa cells untreated or treated with calyculin A, using Phospho-MSK1 (Thr581) Antibody.

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IHC-P (paraffin) Image 13

Immunohistochemical analysis of paraffin-embedded human lung carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb.

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IHC-P (paraffin) Image 14

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with TPA #4174 (right), using Phospho-p90RSK (Ser380) (D3H11) Rabbit mAb.

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IHC-P (paraffin) Image 15

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization, using Phospho-MSK1 (Thr581) Antibody.

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IHC-P (paraffin) Image 16

Immunohistochemical analysis using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb on SignalSlide™ Phospho-p44/42 MAPK (Thr202/Tyr204) IHC Controls #8103 (paraffin-embedded NIH/3T3 cells, treated with U0126 #9903 (left) or TPA #4174 (right).

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IF-IC Image 17

Confocal immunofluorescent analysis of NIH/3T3 cells, serum-starved (left), treated with TPA #4174 (200 nM, 15 min; center), or treated with TPA followed by λ phosphatase (right), using Phospho-p90RSK (Ser380) (D3H11) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Flow Cytometry Image 18

Flow cytometric analysis of Jurkat cells, treated with U0126 (10uM, 2 hrs; blue) or treated with TPA #4174 (200nM, 30 min; green) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412.

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IF-IC Image 19

Confocal immunofluorescent analysis of Drosophila egg chambers, untreated (top) or λ phosphatase-treated (bottom), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and S6 Ribosomal Protein (54D2) Mouse mAb #2317 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 20

Confocal immunofluorescent analysis of HT1080 cells, starved overnight then treated with U0126 #9903 (10 uM, 2 h; left) or PDBu (Phorbol 12,13-Dibutyrate) #12808 (100 nM, 15 m; right) using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-c-Raf (Ser338) (56A6) Rabbit mAb 9427 20 µl
  • WB
H M R Mk 74 Rabbit IgG
Phospho-MEK1/2 (Ser217/221) (41G9) Rabbit mAb 9154 20 µl
  • WB
  • IP
H M R Mk Dm 45 Rabbit 
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb 4370 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Hm Mk Mi Dm Z B Dg Pg Sc 44, 42 Rabbit IgG
Phospho-p90RSK (Ser380) (D3H11) Rabbit mAb 11989 20 µl
  • WB
  • IHC
  • IF
H M R Mk Mi 90 Rabbit IgG
Phospho-MSK1 (Thr581) Antibody 9595 20 µl
  • WB
  • IP
  • IHC
H M 90 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Phospho-Erk1/2 Pathway Sampler Kit provides an economical means of evaluating multiple members of the Erk pathway as well as their activation state. The kit contains enough primary and secondary antibodies to perform two Western blot experiments.

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

Polyclonal and monoclonal antibodies are produced by immunizing rabbits with synthetic phosphopeptides 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 Thr581 of human MSK1. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

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 research investigators consider it 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.
Entrez-Gene Id
5894 , 5595 , 5594 , 5604 , 5605 , 9252 , 6195 , 6197 , 6196
Swiss-Prot Acc.
P04049 , P27361 , P28482 , Q02750 , P36507 , O75582 , Q15418 , P51812 , Q15349
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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