Fig. 6. mAKAP-bound PKA is important for the transduction of signals that induce cardiac myocyte hypertrophy. (A-C) Following co-transfection with expression vectors for GFP and mAKAP siRNA (a-d), primary myocytes were cultured for 2 days in minimal medium containing either no drug (A), 10 µmol/l Iso (B), or 100 µmol/l PE (C). Additional myocytes were co-transfected with a third expression plasmid for either myc-tagged mAKAP WT (e-h) or mAKAP Del PKA BD (i-l). Cells were stained with myc-tag (blue, b,f,j) and mAKAP VO56 (red, c,g,k) antibodies. The nuclei of transfected, GFP-expressing myocytes (green) are marked with arrowheads on the mAKAP and myc panels. Bar, 20 µm. (D) mAKAP WT and mAKAP Del PKA BD protein were expressed by adenoviral infection of COS-7 cells and purified by immunoprecipitation. PKA binding activity of the WT and mutant protein was assayed by RII overlay assay using recombinant His-tagged RII protein and HRP-conjugated anti-His antibody. Equal loading was demonstrated by immunoblotting the same filter with VO54 mAKAP antibody (n=3). (E) Mean cell surface area (±s.e.m.) for myocytes cultured as in A. ^*P<0.015, ^**P<0.0001 for the samples compared; n>115 from 6 independent cultures; ANOVA, P<10^-4 for the entire data set, and Iso- and PE-treated data alone.