HeLa cells growing in medium containing serum were serum starved for 12 hours and stimulated with forskolin. Where indicated, cells were incubated with H-89 (10 μM), LY 294002 (LY, 100 μM), rapamycin (Ra, 100 nM) or U0126 (U, 10 μM) for 1 hour before stimulation with forskolin (20 μM) for a further 30 min. PDE3A was immunoprecipitated (IP) from 1.5 mg of each cell extract with 10 μg anti-PDE3A (1095 – 1110). The immunoprecipitates were, subjected to SDS-PAGE, transferred to a PVDF membrane and immunoblotted with anti-PDE3A (1095 – 1110) [lower panel] or anti-PDE3A phospho Ser 312 [upper panel]. Binding of the primary antibody was detected using rabbit peroxidase conjugated anti-sheep IgG antibody (1:10.000 dilution, Pierce) followed by enhanced chemiluminescence (ECL, Amersham).