pCMV-Raf-1

Investigate the activity and interactions of key signal transduction pathways with our Dominant-Negative Vector Sets. Each vector sets includes a vector that expresses a strong wild-type protein and one that provides a dominant negative, nonfunctional version—allowing you to either activate or “knock down” the pathway. To set up a complete assay system, you can co-express these vectors with an appropriate cis-acting reporter vector to measure the activation of a signal transduction pathway and quantify differences in activation.

载体特征
Simplify the complexities of signal transduction in vivo
Dissect the network of signal transduction pathways
Screen for compounds that activate key pathways
Combine with cis reporter vectors to create a complete assay system

应用
Measure and quantify signal transduction pathway activity
Activation and knockdown studies

Ras & Raf
The Ras Dominant-Negative Vector Set lets you study Ras, a GTP-binding protein that relays signals affecting many cellular processes, including human tumor development (1). We also offer a vector set for studying Raf, the most highly characterized downstream effector of Ras. You can combine the Ras or Raf Vector Sets with our Pathway Profiling Systems to easily monitor cross-talk between different signaling pathways.

Raf-1, a serine/threonine protein kinase, acts as an intermediate link between upstream and downstream kinases in response to various growth factors and mitogens (1, 2). The activation of Raf-MAP (mitogen-activated protein) kinase cascade is a critical step in cellular transformation induced by oncogenic Ras (3, 4); however, the mechanisms by which Ras mediates Raf-1 activation is unclear. The Raf Dominant-Negative Vector Set is a convenient tool for studying mechanisms that affect Raf-1 regulation. The set consists of three vectors:
 pCMV-Raf-1 Vector—expresses the human, wild-type (wt) v-Raf-1 protein.
 pCMV-RafCAAX Vector—expresses a constitutively active form of the Raf protein. This vector encodes K-ras carboxyl-terminal localization signals, which are targeted to the plasma membrane when RafCAAX is expressed in cells. Studies have shown that Ras activation of Raf involves the recruitment of Raf to the plasma membrane where a separate Ras-independent activation of Raf occurs (5–7). Thus, when RafCAAX is expressed in cells, it is enzymatically active, independent of Ras activation.
 pCMV-RafS621A Vector—expresses a dominant-negative form of the Raf protein that blocks Raf pathway activation. RafS621A protein contains a serine-to-alanine mutation at amino acid 621, altering the phosphorylation site for Raf activation, and therefore, blocking phosphorylation and activation of Raf (2).These proteins are expressed at high levels from the constitutive CMV promoter. The SV40 polyadenylation sequence directs proper processing of the 3' end of the mRNAs. The vector backbone contains an SV40 origin for replication in mammalian cells expressing the SV40 T antigen. A neomycin-resistance cassette (Neor)—consisting of the SV40 early promoter, the Tn5 neomycin/kanamycin resistance gene, and polyadenylation signals from the Herpes simplex virus thymidine kinase (HSV TK) gene—allows kanamycin selection in E. coli and neomycin selection in eukaryotic cells. The vector backbone also provides a pUC origin of replication for propagation in E. coli and an f1 origin for single-stranded DNA production.

Use
These vectors can be transfected into mammalian cells using any standard method. Stable transformants can be selected using G418 (8).
The Raf Dominant-Negative Vector Set can be used with our cis-acting reporter vectors, such as pSRE-SEAP (Cat. No. 631901); this combination allows you to set up a complete assay system to measure differences in activation of the Raf-1 pathway.

Propagation in E. coli
 Suitable host strains: DH5α, HB101 and other general purpose strains. Single-stranded DNA production requires a host containing an F plasmid such as JM101 or XL1-Blue.
 Selectable marker: plasmid confers resistance to kanamycin (50 μg/ml) in E. coli hosts.
 E. coli replication origin: pUC
 Copy number: ~500
 Plasmid incompatibility group: pMB1/ColE1