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Applications: Monitor TGFβ signaling pathway activity. Screen activators or inhibitors of TGFβ/ SMAD signaling pathway. Study effects of RNAi or gene overexpression on the activity of TGFβ pathway.
Background: The transforming growth factor beta (TGFβ) signaling pathway is involved in a diverse range of cell processes such as differentiation, cell cycle arrest, and immune regulation. TGFβ signaling has been linked to cardiac disease, cancer, Alzheimer's and other human diseases. TGFβ proteins bind to receptors on the cell surface, initiating a signaling cascade that leads to phosphorylation and activation of SMAD2 and SMAD3, which then form a complex with SMAD4. The SMAD complex then translocates to the nucleus and binds to the SMAD binding element (SBE) in the nucleus, leading to transcription and expression of TGFβ/ SMAD responsive genes.
Concentration: Both the Reporter and Negative Control Reporter are provided at 60 ng DNA/µl (500 µl each)
Description: The SBE Transient Pack is designed to provide the tools necessary for transiently transfecting and monitoring the activity of TGFβ/SMAD signaling pathway in the cultured cells. The kit contains transfection-ready vectors containing firefly luciferase as a TGFβ/SMAD pathway-responsive reporter and constitutively expressing Renilla luciferase as a transfection control. It also includes the Dual Luciferase detection reagents to detect both luciferase activities and specialized medium for growing and assaying HEK293 cells.The key to the SBE Transient Pack is the SBE luciferase reporter vector, which is a TGFβ pathway-responsive reporter. This reporter contains a firefly luciferase gene under the control of multimerized SBE responsive element located upstream of a minimal promoter. The SBE reporter is premixed with constitutively expressing Renilla luciferase vector that serves as internal control for transfection efficiency.The pack also includes a non-inducible firefly luciferase vector premixed with constitutively expressing Renilla luciferase vector as negative control. The non-inducible luciferase vector contains a firefly luciferase gene under the control of a minimal promoter, without any additional response elements. The negative control is critical to determining pathway-specific effects and background luciferase activity.Additionally, the pack includes cell culture medium (BPS Medium 1) that has been optimized for use with HEK293 cells*. BPS Medium 1 includes MEM medium, 10% fetal bovine serum, 1% non-essential amino acids, sodium pyruvate, and 1% Pen/Strep. Finally, the pack provides the Dual Luciferase (Firefly-Renilla) Assay System. These luciferase reagents provide highly sensitive, stable detection of firefly luciferase activity and Renilla luciferase activity. The dual luciferase reagents can be used directly in cells in growth medium, and can be detected with any luminometer; automated injectors are not required. *Note: the kit may be used with other cell lines than HEK293, but an alternate cell culture medium may be required for optimal cell growth.
Storage Stability: Stable at least 12 months from date of receipt, when stored as directed (-20°C)
Warnings: Avoid freeze/thaw cycles.
Biosafety Level: Not applicable (BSL-1)
References: Moustakas A et al. (2001) Smad regulation in TGF-beta signal transduction. J. Cell Science. 114(Pt 24): 4359-69.
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