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Fig. 1. The mAKAP complex includes the PKA substrate RyR2 and CaNAß. (A) Myocyte cultures were treated for 30 minutes with 1 mmol/l diBu-cAMP or 10 µmol/l Iso. mAKAP-associated RyR2 co-immunoprecipitated with VO54 mAKAP serum (lanes 2-4), but not preimmune control serum (lane 1). The immunoprecipitates were back-phosphorylated with recombinant PKA catalytic subunit and [{gamma}-32P]ATP (top panel). RyR2 was detected by immunoblotting with a RyR2 monoclonal antibody (bottom panel). The bar chart shows relative back-phosphorylation (mean ± s.e.m.). *P<0.003 compared to control; n=5; ANOVA, P<0.001. (B) Immune complexes were precipitated from adult rat heart extracts with non-specific mouse IgG (lane 1), mAKAP monoclonal antibody 720 (lane 2), and goat anti-CaNAß (lane 3). CaNAß (top panel), nesprin-1{alpha} (middle panel) and mAKAP (bottom panel) were detected by immunoblotting. Relative molecular mass markers are indicated. (C) Immune complexes were precipitated from adult rat heart extracts with non-specific goat IgG (lane 1) and goat anti-CaNAß antibody (lane 2). mAKAP (top panel), RyR2 (middle panel) and CaNAß (bottom panel) were detected by immunoblotting. n=3.