Figure 2.
Immunoblot, immunoprecipitation, and cAMP-agarose copurification analyses of AKAP100 in the rat heart. In A, human (lane 2) and rat (lane 3) left ventricle homogenates were separated on a 10% SDS-PAGE gel (100 μg/lane) followed by immunoblotting with anti-AKAP100 antibodies. Unstained molecular weight markers were loaded on lane 1. In B, rat left ventricle homogenates (0.5 ml) were incubated with 10 μg of AKAP100-specific rabbit IgGs (lane 1), 10 μg of RII-specific goat IgGs (lane 2), and 200 μl of cAMP-agarose (lane 3). The AKAP100-IgG complexes were precipitated by incubation with Protein G Sepharose beads. The precipitated proteins were solubilized from the agarose or sepharose beads with SDS-PAGE sample buffer followed by immunoblotting with anti-AKAP100 antibodies. As controls for the assay, 2 μg of rabbit IgGs and goat IgGs were loaded on lanes 4 and 5, respectively. Prestained molecular weight markers are loaded on lane 6. In C, human (lane 1) and rat (lane 2) left ventricle homogenates and human AKAP100 recombinant bacterial lysates (lane 3) were separated on a 10% SDS-PAGE gel (100 μg/lane) followed by immunoblotting with anti-AKAP100 antibodies that have been previously preabsorbed by AKAP100 recombinant bacterial lysates. As a control, 2 μg of rabbit IgGs were loaded on lane 4. In D, AKAP100 recombinant bacterial lysates (50 μg/lane) were separated on a 10% SDS-PAGE gel followed by immunoblotting with affinity-purified anti-AKAP100 IgGs as described in the text. The Hoefer (A–C) and Bio-Rad (D) SDS-PAGE systems were used for these studies. A and B represent different lanes from the same gel. The protein bands in A–C were visualized by color development, whereas the immunoblot shown in D was developed by ECL. Molecular weight markers are in kD.