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To Purchase # 9230S

9230S 1 Kit (10 western blots)

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Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated (-) or treated with TPA #4174 (200 nM, 15 min; +), using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb (upper), MKK3 (D4C3) Rabbit mAb #8535 (middle), or MKK6 (D31D1) Rabbit mAb #8550 (lower).

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Western blot analysis of extracts from various cell lines using MKK3 (D4C3) Rabbit mAb.

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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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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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Immunoprecipitation of phospho-MKK3 (Ser189)/MKK6 (Ser207) from HeLa cells, untreated or treated with TPA #4174 (200 nM, 15 min), using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb (lanes 3 and 4) or Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lanes 5 and 6). Lanes 1 and 2 are 10% input. Western blot analysis was performed using

Phospho-MKK3 (Ser189)/MKK6 (Ser207) Antibody #9231. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.

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Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (40 mJ/cm2 with 30 min recovery; right), using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Image
Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb 12280 100 µl
Western Blotting Immunoprecipitation Immunofluorescence
H M R Mk 38 kDa MKK6, 40 kDa MKK3 Rabbit IgG
MKK3 (D4C3) Rabbit mAb 8535 100 µl
Western Blotting Immunoprecipitation
H M R Mk 38-40 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
Western Blotting
All Goat 
Anti-biotin, HRP-linked Antibody 7075 100 µl
Goat 
20X LumiGLO® Reagent and 20X Peroxide 7003 5 ml each
Western Blotting
 
Biotinylated Protein Ladder Detection Pack 7727 100 µl
 
MKK3/MKK6 Control Cell Extracts 9233 200 µl
 

Product Description

The PhosphoPlus® MKK3/MKK6 (Ser189/207) Antibody Kit provides reagents and protocols for the rapid analysis of the phosphorylation status of MKK3/MKK6 at Ser189/207.


Specificity / Sensitivity

Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb recognizes endogenous levels of MKK3 and MKK6 proteins only when phosphorylated at Ser189 (MKK3) or Ser207 (MKK6). MKK3 (D4C3) Rabbit mAb recognizes endogenous levels of total MKK3 protein. This antibody cross-reacts slightly with MKK6 but demonstrates an approximate 10-fold preference for MKK3 using recombinant, purified proteins.


Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser189 of human MKK3 protein and Ser207 of human MKK6 protein and with a synthetic peptide corresponding to residues near the carboxy terminus of human MKK3 protein.

MKK3 and MKK6 are two closely related dual-specificity protein kinases that activate p38 MAP kinase (1-5). MKK3 and MKK6 both phosphorylate and activate p38 MAP kinase at its activation site, Thr-Gly-Tyr, but do not phosphorylate or activate Erk1/2 or SAPK/JNK. Phosphorylation of p38 MAP kinase dramatically stimulates its ability to phosphorylate protein substrates such as ATF-2 and Elk-1. MKK3 and MKK6 are both activated by different forms of cellular stress and inflammatory cytokines (4,5). Activation of MKK3 and MKK6 occurs through phosphorylation at Ser189 and Thr222 on MKK3 (2) and Ser207 and Thr211 on MKK6 (4,5).


1.  Derijard, B. et al. (1995) Science 267, 682-685.

2.  Raingeaud, J. et al. (1995) J. Biol. Chem. 270, 7420-7426.

3.  Sluss, H.K. et al. (1994) Mol. Cell. Biol. 14, 8376-8384.

4.  Raingeaud, J. et al. (1996) Mol. Cell. Biol. 16(3), 1247-1255.

5.  Han, J. et al. (1996) J. Biol. Chem. 271, 2886-2891.


Entrez-Gene Id 5606
Swiss-Prot Acc. P46734


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
PhosphoPlus® is a trademark of Cell Signaling Technology, Inc.
LumiGLO® is a registered trademark of Kirkegaard & Perry Laboratories.