Cell Signaling Technology

Product Pathways - MAPK Signaling

c-Raf (D4B3J) Rabbit mAb #53745

No. Size Price
53745S 100 µl ( 10 western blots ) ¥3,100.00 现货查询 购买询价
53745 carrier free & custom formulation / quantityemail request
Applications Dilution Species-Reactivity Sensitivity MW (kDa) Isotype
W 1:1000 Human,Mouse,Rat, Endogenous 75 Rabbit IgG
IP 1:50

Species cross-reactivity is determined by western blot.

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

Specificity / Sensitivity

c-Raf (D4B3J) Rabbit mAb recognizes endogenous levels of total c-Raf protein.

Source / Purification

Monoclonal antibody is produced by immunizing animals with recombinant protein specific with a central region of human c-Raf protein.

Western Blotting

Western Blotting

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human c-Raf (hc-Raf-Myc/DDK; +) using c-Raf (D4B3J) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from various cell lines using c-Raf (D4B3J) Rabbit mAb.

Western Blotting

Western Blotting

Western blot analysis of extracts from HT-29 cells, untreated (-) or treated with 17-AAG #8132 (1 μM, 24 hrs; +) using c-Raf (D4B3J) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

IP

IP

Immunoprecipitation of c-Raf from KARPAS-299 cell extracts. Lane 1 represents 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is c-Raf (D4B3J) Rabbit mAb. Western blot was performed using c-Raf (D4B3J) Rabbit mAb. A conformation specific secondary antibody was used to avoid cross reactivity with IgG.

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, 279-83.
  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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Protocols

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

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

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

KARPAS cell line source: Dr. Abraham Karpas at the University of Cambridge.

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

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