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Fig. 5. Phosphorylated RII{alpha} binds with different affinity to the RII-binding motifs in AKAP450. Purified recombinant human RII{alpha} was radiolabeled using the catalytic subunit of either PKA (A) or CDK1 (B) and used for overlays on blots with different AKAPs (100 ng). Lanes: 1, GST-AKAP79 (178-427); 2, GST-AKAP149 (285-387); 3, GST-AKAP450 (1390-1595); 4, GST-AKAP450 (2327-2602) (numbers indicate the amino acid extent of the recombinant fragments used). (C) Precipitation of RII{alpha} isoforms with different GST-AKAPs (20 nM of both R and AKAP). In some experiments, RII{alpha} was preincubated with CDK1 before the GST precipitation. Lanes: 1, RII{alpha} (50 ng); 2, RII{alpha} after CDK1 preincubation; 3, GST + RII{alpha} (negative control); 4, GST-AKAP450 (1390-1595) + RII{alpha}; 5, GST-AKAP450 (1390-1595) + RII{alpha}-P; 6, GST-AKAP450 (2327-2602) + RII{alpha}; 7, GST-AKAP450 (2327-2602) + RII{alpha}-P; 8, GST-AKAP79 (178-427) + RII{alpha}; 9, GST-AKAP149 (285-387) + RII{alpha}. Immunodetection was with anti-hRII{alpha} mAb.