PDE4D Cell-Based Activity BioAssay™ Kit

Kits and Assays / Kits-Molecular Biology, BioAssay™

-20°C/-70°C

Phosphodiesterases (PDEs) play an important role in the dynamic regulation of cAMP and cGMP signaling. PDE4D has 3', 5'-cyclic-AMP phosphodiesterase activity and degrades cAMP. Inhibition of PDE4D activity by its inhibitors leads to an elevated intracellular level of cAMP. The PDE4D gene encodes at least 9 different isoforms, and has been linked to stroke, asthma, arrhythmia, and cardiac myopathy, making it an important therapeutic target. The PDE4D cell-based activity BioAssay™ is designed for screening inhibitors of PDE4D7 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 PDE4D7 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 PDE4D inhibitor to inhibit PDE4D7 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), PDE4D7 expression vector, and forskolin.

Applications:
Screen PDE4D inhibitors for drug discovery.
Monitor cAMP/PDE4D signaling pathway activity.

Components:
Reporter (Component A), CRE luciferase reporter vector + constitutively expressing Renilla luciferase vector, 1x500ul (60ng DNA/ul)
PDE4D7 expression vector (Component B), 1x250ul (80ng DNA/ul)
Forskolin, 10mM, in DMSO, 1x25ul

Storage and Stability:
Store powder at 4°C liquid at -20°C. Store other components at 4°C. Stable for at least 6 months For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.