PDE4B Cell-Based Activity Assay Kit

Catalog #: 79526
Size: 500 Reactions
$575
Description:

Phosphodiesterases (PDEs) play an important role in the dynamic regulation of cAMP and cGMP signaling. PDE4B has 3',5'-cyclic-AMP phosphodiesterase activity and degrades cAMP. Inhibition of PDE4B activity by its inhibitors leads to an elevated intracellular level of cAMP. The PDE4B gene encodes at least 4 different isoforms, and has been linked to inflammation in monocytes by its regulation of the toll-receptor pathway. It is also highly expressed in the central nervous system, and has been targeted as a potential treatment for autism, depression, schizophrenia, and other conditions.

The PDE4B cell-based activity assay kit is designed for screening inhibitors of PDE4B1 in cultured cells. The assay is based on transfecting cells with the CRE luciferase reporter. CRE reporter contains the firefly luciferase gene under the control of cAMP response element (CRE). Elevation of intracellular cAMP activates CRE binding protein (CREB) to bind CRE and induce the expression of luciferase. Forskolin is commonly used to raise the intracellular level of cAMP in cell physiology studies. When cells transiently transfected with CRE reporter are activated by forskolin, the intracellular level of cAMP is upregulated, which induces the expression of CRE luciferase reporter. However, when cells are co-transfected with PDE4B1 expression vector and CRE reporter, the level of forskolin-induced cAMP is reduced, resulting in lower expression level of luciferase. When cells are treated with PDE4B inhibitor to inhibit PDE4B1 activity, cAMP level is restored, resulting in higher luciferase activity.

The kit includes CRE luciferase reporter (premixed with constitutively-expressing Renilla (sea pansy) luciferase vector that serves as an internal control for transfection efficiency), PDE4B1 expression vector, and forskolin.

Materials Required But Not Provided:
• Mammalian cell line and appropriate cell culture medium
• 96-well tissue culture-treated white clear-bottom assay plate (Corning #3610) 
• Transfection reagent for mammalian cell line [We use Lipofectamine™ 2000 (Invitrogen #11668027). However, other transfection reagents work equally well.] 
• Opti-MEM I Reduced Serum Medium (Invitrogen #31985-062) 
• Dual luciferase assay system: Dual Luciferase (Firefly-Renilla) Assay System (BPS Bioscience #60683): This system assays cells directly in growth medium. It can be used with any luminometer. Automated injectors are not required. 
• Luminometer 
Synonym(s):
PDE4B, cAMP-specific 3',5'-cyclic phosphodiesterase 4B, phosphodiesterase E4 dunce homolog (Drosophila), phosphodiesterase 4B
Format:

COMPONENTS:

Instructions for use:
See assay kit data sheet for detailed protocol.
Storage / Stability:
Stable at least 6 months from date of receipt, when stores as directed. Kit components require different storage conditions. Be sure to store each component at the proper temperature upon arrival.
Application(s):
Screen PDE4B inhibitors for drug discovery and monitor cAMP/PDE4B signaling pathway activity.
Reference(s):

Campbell, Susan L. et al. 2017. “Altered Phosphorylation, Electrophysiology, and Behavior on Attenuation of PDE4B Action in Hippocampus.” BMC Neuroscience 18: 77.

Fox, David, Alex B. Burgin, and Mark E. Gurney. 2014. “Structural Basis for the Design of Selective Phosphodiesterase 4B Inhibitors.” Cellular Signaling 26 (3): 657–663. PMC. Web. 14 Aug. 2018.

Zhang, Chong et al. 2017. “Comparison of the Pharmacological Profiles of Selective PDE4B and PDE4D Inhibitors in the Central Nervous System.” Scientific Reports 7: 40115.

Warning(s):

Avoid freeze/thaw cycles.

Scientific Category:
Phosphodiesterase
Product Type:
Assay Kit
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Product Datasheets

79526.pdf