Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-c-Raf (Ser296) Antibody #9423

Applications Reactivity Sensitivity MW (kDa) Source
W H M R Endogenous 74 Rabbit

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

Specificity / Sensitivity

Phospho-c-Raf (Ser296) Antibody detects endogenous levels of c-Raf protein only when phosphorylated at Ser296.

Source / Purification

Polyclonal antibodies are produced by immunizing rabbits with a synthetic phospho-peptide (KLH-coupled) corresponding to residues surrounding Ser296 of human c-Raf. Antibodies are purified by protein A and peptide affinity chromatography.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293 and NIH/3T3 cells, untreated or treated with TPA + FBS, using Phospho-c-Raf (Ser296) Antibody.

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.

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This product is for in vitro research use only and is not intended for use in humans or animals. This product is not intended for use as therapeutic or in diagnostic procedures.

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