Quantitation of Intracellular cAMP

To quantify intracellular cAMP concentrations, the cAMP-Screen Cyclic AMP Immunoassay System (ThermoFisher, USA) was utilized following the manufacturer’s protocol. Briefly, cell lysates from treated and non-treated cells were incubated with a cAMP-Alkaline phosphatase conjugate (cAMP-AP) and an anti-cAMP antibody in a coated microtiter plate. In the absence of intracellular cAMP, all cAMP-AP conjugate is captured on the coated surface, resulting in a high signal. When present in the cell lysate, intracellular cAMP competes with the cAMP-AP causing a reduced signal with signal reduction being proportional to the amount of cAMP present in the cell lysate. After washing to remove unbound cAMP-AP, the chemiluminescent alkaline phosphatase substrate is added, and the resulting glow signal is measured using a luminometer.