| 货号 | 8935S |
| 反应种属 | Human |
| 应用 | TFN |
| 使用方法 | CST recommends transfection with 100 nM SignalSilence® B-Raf siRNA I 48 to 72 hours prior to cell lysis. For transfection procedure, follow protocol provided by the transfection reagent manufacturer. Please feel free to contact CST with any questions on use. Each vial contains the equivalent of 100 transfections, which corresponds to a final siRNA concentration of 100 nM per transfection in a 24-well plate with a total volume of 300 µl per well. |
| 供应商 | CST |
| 背景 | 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). |
| 存放说明 | -20C |
| 参考文献 | 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. |
Western blot analysis of extracts from HT-29 cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® B-Raf siRNA I (+), or SignalSilence® B-Raf siRNA II #9439 (+), using B-Raf (55C6) Rabbit mAb #9433 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). The B-Raf (55C6) Rabbit mAb confirms silencing of B-Raf expression, while the β-Actin (D6A8) Rabbit mAb is used as a loading control.转染了100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® B-Raf siRNA I (+), 或SignalSilence® B-Raf siRNA II #9439 (+)的HT-29细胞提取物，使用B-Raf (55C6) Rabbit mAb #9433（上）或β-Actin (D6A8) Rabbit mAb #8457（下）进行western blot分析。B-Raf (55C6) Rabbit mAb用以确认B-Raf的敲除，β-Actin (D6A8) Rabbit mAb则作为上样对照。 |
