Product Pathways - MAPK Signaling
c-Raf (D4B3J) Rabbit mAb #53745
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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 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 blot analysis of extracts from various cell lines using c-Raf (D4B3J) Rabbit mAb.
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).
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.
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).
- Avruch, J. et al. (1994) Trends Biochem Sci 19, 279-83.
- Chong, H. et al. (2001) EMBO J 20, 3716-27.
- King, A.J. et al. (1998) Nature 396, 180-3.
- Fabian, J.R. et al. (1993) Mol Cell Biol 13, 7170-9.
- Mason, C.S. et al. (1999) EMBO J 18, 2137-48.
- Zimmermann, S. and Moelling, K. (1999) Science 286, 1741-4.
- Sprenkle, A.B. et al. (1997) FEBS Lett 403, 254-8.
- Marais, R. et al. (1997) J Biol Chem 272, 4378-83.
- Guan, K.L. et al. (2000) J Biol Chem 275, 27354-9.
- Davies, H. et al. (2002) Nature 417, 949-54.
- Dougherty, M.K. et al. (2005) Mol Cell 17, 215-24.
Have you published research involving the use of our products? If so we'd love to hear about it. Please let us know!
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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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