Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb #12280

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP IF-IC H M R Mk (Z) (B) (GP) Endogenous 38 kDa MKK6, 40 kDa MKK3 Rabbit IgG

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  IF-IC=Immunofluorescence (Immunocytochemistry)
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Z=Zebrafish  B=Bovine  GP=Guinea Pig
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

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).

Source / Purification

Monoclonal antibody is produced by immunizing animals with synthetic peptides corresponding to residues surrounding Ser189 of human MKK3 protein and Ser207 of human MKK6 protein.

Western Blotting

Western Blotting

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).

IP

IP

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

IF-IC

IF-IC

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).


Background

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.

Application References

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

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