Fig. 2. Biochemical characterization of the mAKAP signaling complex. The mAKAP signaling complex was analyzed by a series of complementary biochemical approaches. (A) A schematic diagram depicting the isolation of the mAKAP immune complexes. (B) Immunoprecipitations were performed from rat heart extracts using antibodies against rat mAKAP or control IgG. The resulting immune complexes were separated by electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. Immunoblot analyses using monoclonal antibodies against the PDE4D family were used to identify the PDE in heart extracts (lane 1) and immunoprecipitations with the IgG control (lane 2) or anti-mAKAP antisera (lane 3). Detection of signals was by chemiluminescence. Molecular weight markers are indicated. The migration of PDE4D is indicated. (C) A schematic diagram depicting immunoprecipitation of PDE4D and associated proteins. (D) Immune complexes were isolated from rat heart extracts (lane 1) using goat polyclonal antibodies against PDE4D isoforms (lane 3) or control goat IgG (lane 2). Immune complexes were separated by electrophoresis on a 7.5% SDS–polyacrylamide gel and electrotransferred to nitrocellulose membranes. The filter was subjected to immunoblot analysis using rabbit polyclonal antibodies against rat mAKAP (top panel), RII regulatory subunit (middle panel) and catalytic subunit of PKA (bottom panel). Detection of signals was by chemiluminescence. Molecular weight markers and the migration position of mAKAP are indicated. (E) PKA activity in PDE4D immune complexes was measured using the heptapeptide Kemptide as a substrate. PKA specific activity (pmol/min/IP) was measured from PDE4D immune complexes, the IgG control and in the presence of the PKI 5–24 peptide, a specific inhibitor of PKA. The source of the sample is indicated below each lane. The accumulated data from three experiments are presented.