Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-A-Raf (Ser299) Antibody #4431


No. Size Price
4431S 100 µl ( 10 western blots ) ¥3,900.00 现货查询 购买询价
4431T 20 µl ( 2 western blots ) ¥1,500.00 现货查询 购买询价
4431 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat, Endogenous 68 Rabbit

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting,

Specificity / Sensitivity

Phospho-A-Raf (Ser299) Antibody detects endogenous levels of A-Raf only when phosphorylated at serine 299. The antibody does not cross-react with B-Raf or Raf-1 under physiological conditions.

Phospho-A-Raf (Ser299) 兔多抗能检测内源性丝氨酸(229位点)磷酸化的A-Raf蛋白水平。这种抗体在生理条件下不会与c-Raf 和 B-Raf蛋白发生交叉反应。

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser299 of human A-Raf. Antibodies are purified by protein A and peptide affinity chromatography.


Western Blotting

Western Blotting

Western blot analysis of extracts from Raji and HeLa cells, untreated or TPA-treated (30 minutes), using Phospho-A-Raf (Ser299) Antibody (upper) or A-Raf Antibody, #4432 (lower).western blot方法检测细胞提取物:未经处理和TPA处理(30分钟)的Raji 、 HeLa细胞,使用的抗体是Phospho-A-Raf (Ser299) Antibody (上图) 和A-Raf Antibody, #4432 (下图)。


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).

A-Raf, B-Raf, c-Raf (Raf-1)是由GTP结合蛋白-Ras蛋白所募集的主要效应因子,能激活MEK-MAPK途径(1)。目前对c-Raf的活化机制了解的最清楚,它涉及到许多活化位点的磷酸化,包括Ser338,Tyr341,Thr491,Ser494,Ser497和Ser499位点(2)。p21活化的蛋白激酶(PAK)能磷酸化c-Raf蛋白的Ser338位点 ,Src家族蛋白能磷酸化c-Raf蛋白的Tyr341位点,进而诱导c-Raf的活化(3,4)。c-Raf蛋白的Ser338位点,与A-Raf 的Ser299位点 、 B-Raf 的Ser445位点相对应,尽管B-Raf蛋白的Ser445位点已经被组成性的磷酸化 (5)。Akt和AMPK可以分别磷酸化c-Raf蛋白的Ser259和Ser621位点,从而抑制14-3-3与c-Raf的结合(6,7)。虽然A-Raf, B-Raf, c-Raf的序列和功能相似,但是调节方式各不相同(8)。值得关注的是,B-Raf蛋白包含三个Akt的磷酸化位点(Ser364,Ser428和Thr439位点),但缺少与c-Raf蛋白的Tyr341位点相对应的磷酸化位点(8,9)。研究表明,B-Raf蛋白的突变体V600E,会导致激酶活性的调高,而且这种突变在恶性黑色素瘤中普遍存在(10)。c-Raf蛋白的六个位点 (Ser29, Ser43, Ser289, Ser296, Ser301和Ser642位点)会以某种方式过度磷酸化,这与c-Raf蛋白的失活有关。这六个位点的过度磷酸化依赖于下游的MEK信号转导,并使c-Raf不应答后续的活化事件(11)。

  1. Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
  2. Chong, H. et al. (2001) EMBO J 20, 3716-27.
  3. King, A.J. et al. (1998) Nature 396, 180-3.
  4. Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
  5. Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
  6. Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
  7. Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
  8. Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
  9. Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
  10. Davies, H. et al. (2002) Nature 417, 949-54.
  11. Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.

Application References

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