Cell Signaling Technology

Product Pathways - MAPK Signaling

c-Raf Antibody #9422

Applications Reactivity Sensitivity MW (kDa) Source
W H M R Mk Endogenous 65 to 75 Rabbit

Applications Key:  W=Western Blotting
Reactivity Key:  H=Human  M=Mouse  R=Rat  Mk=Monkey
Species cross-reactivity is determined by western blot. Species enclosed in parentheses are predicted to react based on 100% sequence homology.

Protocols

Specificity / Sensitivity

c-Raf Antibody detects endogenous levels of total c-Raf protein.

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Pro302 of human c-Raf. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa, C2C12 or PC12 cells, untreated or TPA-treated (200 nM for 30 minutes), using c-Raf Antibody #9422.

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). Research studies have shown that 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

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

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

Products