After removing the supernatant, cells were washed three times in cold phosphate-buffered saline and resuspended in 500 μl of cell lysis buffer. Cells were frozen at −20°C and thawed with gentle mixing. The freeze/thaw cycle was repeated three times, and the mixture was centrifuged at 600 g for 10 min at 2-8°C to remove cellular debris. The supernatant was collected and stored at −20°C. cAMP concentrations were then measured using the cyclic adenosine monophosphate (cAMP) Enzyme Immuno-Assay (EIA) kit (KGE002B, R & D systems, USA).