Cell Signaling Technology

Product Pathways - MAPK Signaling

Phospho-c-Raf (Ser259) Antibody #9421

a-Raf   c-raf   cRaf   Raf-1   Raf1   rasraf  

No. Size Price
9421S 100 µl ( 10 western blots ) ¥3,900.00 现货查询 购买询价
9421T 20 µl ( 2 western blots ) ¥1,500.00 现货查询 购买询价
9421 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat,Monkey,Xenopus, Endogenous 74 Rabbit
IP 1:50

Species cross-reactivity is determined by western blot.

Applications Key: W=Western Blotting, IP=Immunoprecipitation,

Homology

Species predicted to react based on 100% sequence homology: Chicken,

Specificity / Sensitivity

Phospho-c-Raf (Ser259) Antibody detects endogenous levels of c-Raf only when phosphorylated at Ser259.

Phospho-c-Raf (Ser259) Antibody兔多抗能检测内源性丝氨酸(259位点)磷酸化的c-Raf蛋白水平。

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser259 of human c-Raf. Antibodies are purified by protein A and peptide affinity chromatography.

该多克隆抗体通过使用与人源 c-Raf蛋白丝氨酸(259位点)周围残基相一致的合成磷酸化肽段免疫动物而获得。该抗体经蛋白A和肽亲和层析纯化。

Western Blotting

Western Blotting

Site specificity of Phospho-c-Raf (Ser259) Antibody: Western blot analysis of recombinant Myc-tagged c-Raf protein, wild-type (lanes 1 and 3) and S259A mutant (lanes 2 and 4), using Phospho-Raf (Ser259) Antibody or a Myc antibody. (Provided by Dr. Guri Tzivion, Massachusetts General Hospital.)Phospho-c-Raf (Ser259) Antibody位点特异性:Western blot方法检测Myc标记的c-Raf重组蛋白,野生型(1、3号条带)和S259A 突变体(2、4号条带)。使用的抗体是Phospho-Raf (Ser259) Antibody 和Myc antibody。(由Dr. Guri Tzivion提供, Massachusetts General Hospital)。

Western Blotting

Western Blotting

Western blot analysis of extracts from HeLa cells, untreated or TPA-treated, using Phospho-c-Raf (Ser259) Antibody (upper), or a total c-Raf antibody (lower).Western blot方法检测细胞提取物:未经处理和TPA处理的HeLa细胞。使用的抗体是Phospho-c-Raf (Ser259) Antibody (上图)和a total c-Raf antibody (下图)。

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).

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.

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Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.

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