Fig. 4. mAKAP-RyR complex is specific to the nucleus. (A) Adult rat hearts were fractionated for comparison of the constituents of different membrane compartments. Whole heart homogenate (WH, lane 1) was centrifuged at 3800 g. The resulting pellet (P1, lane 3) contained myofibrils, mitochondria and nuclei. Nuclei (lane 5) were purified from P1 by 2.4 M sucrose step gradient centrifugation at 50,000 g and repeat low-speed centrifugation. The 3800 g supernatant fraction (S1, lane 2) was clarified at 10,000 g, before centrifugation at 100,000 g. The 100,000 g pellet (P2, lane 4) contained sarcoplasmic reticulum, Golgi apparatus and plasma membrane. 5 µg protein for each fraction was subjected to SDS-PAGE and immunoblotting with purified mAKAP antibody and monoclonal RyR antibody. Antibodies for LAP-2, a nuclear matrix protein, and calsequestrin, a SR calcium-binding protein, served as indicators for the quality of the fractionations. (B) Comparison with pure SR. 5 µg whole heart homogenate (WH, lane 1), 25 µg purified nuclei (lane 2), and 25 µg purified sarcoplasmic reticulum (SR, lane 3) were subject to immunoblotting with anti-mAKAP VO54 antibody. SR fraction was prepared from P2 fraction by sucrose gradient centrifugation. (C) mAKAP and RyR are still associated after purification of nuclei. 75 µg of purified heart nuclei were treated as starting material in immunoprecipitation experiments with control preimmune serum (lane 1) or specific mAKAP VO54 antiserum (lane 2), and RyR was detected in immunoprecipitates by immunoblotting as performed in Fig. 2. As assessed by light microscopy, the nuclei used in these experiments remained intact throughout their purification (not shown). The migration of the respective protein and molecular weight markers are indicated for each panel. n=3 for the experiments represented in each panel.