Fig. 2. Rolipram induces protein kinase A activity in REF52 cells. Cells were allowed to adhere to laminin-coated plates in the absence (–) or presence (+) of rolipram before lysis in the appropriate buffer. (A) PKA activity in lysates was assayed using a peptide substrate. Data shown are of three independent experiments (mean±s.e.m.). (B) The level of phosphorylation of the PKA-substrate CREB was determined by western blotting using an antibody specific for CREB phosphorylated at Ser133. An antibody that recognises phosphorylated and unphosphorylated CREB equally well was used to demonstrate equal loading. The PKA selective inhibitor H89 (1 µM) was found to inhibit the rolipram induced phosphorylation of CREB. (C) Western blotting with an antibody that recognises VASP was used to determine the phosphorylation state of this protein as it undergoes a mobility shift when phosphorylated at Ser157. The blot was re-probed with an antibody specific for actin to demonstrate equal loading.