Description:

Principle:

Cyclic AMP response element (CRE)-binding protein (CREB) is a transcription factor that regulates and responds to diverse cellular responses, ranging from proliferation, survival, differentiation, stress responses, and neuronal activity. These cellular signals lead to upstream kinase activation, such as protein kinase A (PKA), pp90 ribosomal S6 kinase (pp90RSK), and Ca2+/calmodulin-dependent protein kinases (CaMKs), and these kinases in turn phosphorylate CREB to induce CREB activity. CREB increases the transcription of genes that contain cAMP responsive elements.

Signosis has developed CREB luciferase reporter stable cell line by co-transfecting CREB luciferase reporter vector and hygromycin expression vector. The hygromycin resistant clones were subsequently screened for forskolin-induced luciferase activity. The cell line can be used as a reporter system for monitoring the activation of CREB triggered by stimuli treatment, such as forskolin and gene overexpression and gene knockdown.

Principle behind TF luciferase reporter. TF luciferase reporter stable cell line utilizes artificial promoter constructs to drive luciferase expression. The promoter region can consists of multiple repeats of a cis-element TF binding site, a DNA fragment from the promoter region of a known TF downstream gene, or a DNA fragment containing putative/known TF binding sites. There are several ways that a TF can be activated, such as through extracellular stimuli or through intracellular signaling pathways. Once activated, the TF translocates to the nucleus and often interacts with relevant co-factors to drive gene expression. Once luciferase is expressed, it can generate light in an enzymatic assay and the amount of light measured is positively correlated with the level of TF activation.

Data:

Analysis of SL-0020 CREB reporter activity in response to forskolin treatment.The cells were seeded on a 96-well plate for overnight with DMEM including 10% FBS. The cells then were treated with or without 10uM forkoslin in DMEM and 0.1% FBS for 16 hours. Forskolin induced over 50-fold increase in luciferase activity compared to untreated cells.