Fig. 3.

Phosphorylation of PDE3A1 and PDE3A2 in response to PKA and PKC activation. HEK293 cells were transfected with FLAG-tagged PDE3A2 and PDE3A1. (A) Before lysis and immunoprecipitation with anti-FLAG antibodies, cells were treated with 1 µM isoproterenol for 90 s or with 10 ng/mL PMA for 15 min. (B) The experiment was carried out in cells incubated in the absence and presence of 100 µM IBMX before treatment with 1 µM isoproterenol for 90 s. (C) FLAG-tagged PDE3A2 and PDE3A1, affinity-purified from transfected cells using anti-FLAG antibodies, were incubated in the absence or presence of 10 or 100 nM PKA and 200 µM ATP for 20 min at 30 °C. Phosphorylation was analyzed by Western blot analysis with phosphospecific antibodies as indicated. The coimmunoprecipitation of endogenous 14-3-3 was analyzed by immunoblotting with anti–14-3-3 antibody.