PDE4D Cell-Based Reporter Assay Kit
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 assay 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.
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• 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)
• Forskolin (Sigma Aldrich #F3917)
• TWO-Step Luciferase (Firefly & Renilla) Assay System:
TWO-Step 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
| Component | Specification | Amount | Storage |
| Reporter (Component A) | CRE luciferase reporter vector + constitutively expressing Renilla luciferase vector |
500 μl (60 ng DNA/ μl) |
-20°C |
| PDE4D7 expression vector (Component B) |
250 μl (40 ng DNA/ μl) |
-20°C |
These vectors are designed for transient transfection. They are NOT suitable for transformation
and amplification in bacteria.
1. Montminy MR et al. (1987). Nature 328(6126):175-178
2. Fan Chung, K. (2006). Eur. J. Pharmacol. 533(1-3):110-117
3. Malik, R. et al. (2008). Appl. Microbiol. Biotechnol. 77(5): 1167-1173
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