HeLa cells growing in medium containing serum were harvested (S), or incubated with mimosine (Mim 0.2 mM for 16 h), aphidicolin (Aph 10 μg/ml for 16 h) or serum-starved for 12 hours and stimulated with PMA (TPA 400 ng/ml or forskolin (FO 20 μM). 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 analysed by DIG-14-3-3 overlay and immunoblotted with antibodies that recognise the pSer 312, pSer 428, pSer 438, pSer 465 and pThr 568 sites on PDE3A. 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).