Cell Signaling Technology

Product Pathways - MAPK Signaling

B-Raf (L12G7) Mouse mAb #9434

Applications Reactivity Sensitivity MW (kDa) Isotype
W IP F H M R Mk Dm Endogenous 86 Mouse IgG1

Applications Key:  W=Western Blotting  IP=Immunoprecipitation  F=Flow Cytometry
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey  Dm=D. melanogaster
Species cross-reactivity is determined by Western blot.

Protocols

Specificity / Sensitivity

B-Raf (9L12G7) Mouse mAb detects endogenous levels of total B-Raf protein.  

Source / Purification

Monoclonal antibody is produced by immunizing animals with GST-partial B-Raf proteins.  

Western Blotting

Western Blotting

Western blot analysis of extracts from K-562, HeLa, C1C12, NBT-II and COS cells, using B-Raf (L12G7) Mouse mAb.

Flow Cytometry

Flow Cytometry

Flow cytometric analysis of K-562 cells, using B-Raf (L12G7) Mouse mAb (blue) compared to a nonspecific negative control antibody (red).

Background

A-Raf, B-Raf and c-Raf (Raf-1) are the main effectors recruited by GTP-bound Ras to activate the MEK-MAP kinase pathway (1). Activation of c-Raf is the best understood and involves phosphorylation at multiple activating sites including Ser338, Tyr341, Thr491, Ser494, Ser497 and Ser499 (2). p21-activated protein kinase (PAK) has been shown to phosphorylate c-Raf at Ser338 and the Src family phosphorylates Tyr341 to induce c-Raf activity (3,4). Ser338 of c-Raf corresponds to similar sites in A-Raf (Ser299) and B-Raf (Ser445), although this site is constitutively phosphorylated in B-Raf (5). Inhibitory 14-3-3 binding sites on c-Raf (Ser259 and Ser621) can be phosphorylated by Akt and AMPK, respectively (6,7). While A-Raf, B-Raf and c-Raf are similar in sequence and function, differential regulation has been observed (8). Of particular interest, B-Raf contains three consensus Akt phosphorylation sites (Ser364, Ser428 and Thr439) and lacks a site equivalent to Tyr341 of c-Raf (8,9). The B-Raf mutation V600E results in elevated kinase activity and is commonly found in malignant melanoma (10). Six residues of c-Raf (Ser29, Ser43, Ser289, Ser296, Ser301 and Ser642) become hyperphosphorylated in a manner consistent with c-Raf inactivation. The hyperphosphorylation of these six sites is dependent on downstream MEK signaling and renders c-Raf unresponsive to subsequent activation events (11).

  1. Avruch, J. et al. (1994) Trends Biochem. Sci. 19, 279-283.
  2. Chong, H. et al. (2001) EMBO J. 20, 3716-3727.
  3. King, A.J. et al. (1998) Nature 396, 180-183.
  4. Fabian, J.R. et al. (1993) Mol. Cell Biol. 13, 7170-7179.
  5. Mason, C.S. et al. (1999) EMBO J. 18, 2137-2148.
  6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-1744.
  7. Sprenkle, A.B. et al. (1997) FEBS Lett. 403, 254-258.
  8. Marais, R. et al. (1997) J. Biol. Chem. 272, 4378-4383.
  9. Guan, K.L. et al. (2000) J. Biol. Chem. 275, 27354-27359.
  10. Davies, H. et al. (2002) Nature 417, 949-954.
  11. Dougherty, M.K. et al. (2005) Mol. Cell 17, 215-224.

Application References

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This product is intended for research purposes only. The product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

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