1. 1 cells were seeded in a 96-well plate at 5 ? 10 4 cells per well, and left to attach overnight. Prior to the experiment, cells were washed with HBSS;J774A
2. SA=115 ?molmin -1 mg -1 ) for 30 min. Finally, the cAMP content was determined by using the CatchPointcAMP immunoassay kit (Molecular Devices, Wokingham, UK). Briefly, the reaction was terminated by the addition of lysis buffer to the treated cells (50 ?L per well), the cellular content was extracted by shaking the plate at 250 rpm for 10 min. The plate was then centrifuged to remove cell debris, the supernatant was transferred to the immunoassay plate, and immunoassays were carried out according to the manufacturer's instructions. Fluorescence signal was acquired using an Infinite M1000 plate reader;HeLa S3, and HepG2 cells. All cell lines were obtained from ATCC
3. Structure of the adenylyl cyclase catalytic core;G Zhang;Nature,1997