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. 2013 Apr 26;288(24):17111–17121. doi: 10.1074/jbc.M112.447326

FIGURE 3.

FIGURE 3.

RSelect subunits exhibit AKAP selectivity in vitro. A, Coomassie Blue-stained SDS-polyacrylamide gel of purified RSelect fragments (residues 1–45). B, RII overlay of a panel of 10 GST-AKAP fusion proteins (Ponceau-stained; first panel) using the D/D domain fragments shown in A: RSelectAKAP2(1–45) (second panel), RSelectAKAP18(1–45) (third panel), RSelectAKAP150(1–45) (fourth panel), and wild-type RII(1–45) (fifth panel). C, AlphaScreen competition binding assay for AKAP18 with D/D domain fragments of either RSelectAKAP18 (red squares) or WT RII (blue triangles) and increasing concentrations of the appropriate untagged R subunit. The Kd (n = 3) for AKAP18 interaction with either RSelectAKAP18 (red squares) or WT RII (blue triangles) is shown. D, competition binding assay for AKAP18 with D/D domain fragments of either RSelectAKAP18 (red squares) or WT RII (blue triangles) in the presence of increasing concentrations of a competitor AKAP peptide mixture. E, the IC50 (n = 3) of the competitor AKAP peptide mixture for AKAP18 associated with either RSelectAKAP18 (red bar) or WT RII (blue bar). F, Coomassie Blue-stained SDS-polyacrylamide gel of the purified full-length RII mutant (RSelectAKAP18FL). G, immunoprecipitations (IP) with HEK293 cell lysates were performed in the presence (lanes 4 and 7) and absence (lanes 1–3, 5, and 6) of RSelectAKAP18FL using IgG (lane 1), anti-V5 antibody (lanes 2–4; for V5-AKAP2), or anti-FLAG antibody (lanes 5–7; for FLAG-AKAP18δ). These were done either with Ht31 (lanes 3, 4, 6, and 7) or without Ht31 (lanes 1, 2, and 5). Anchoring and coprecipitation of the modified PKA holoenzyme were tested by immunoblotting (IB) for the PKA C subunit. Immunoprecipitation of AKAP18δ (anti-FLAG antibody), AKAP2 (anti-V5 antibody), and the PKA C subunit was confirmed by immunoblotting (middle and lower panels). H, AlphaScreen competition binding assay for AKAP2 with either RSelectAKAP2 (red squares) or WT RII (blue triangles) and increasing concentrations of the appropriate untagged R subunit. The Kd (n = 3) for AKAP2 interaction with either RSelectAKAP2 or WT RII is indicted. I, AlphaScreen competition binding assay for AKAP2 with either RSelectAKAP2 (red squares) or WT RII (blue triangles) in the presence of increasing concentrations of a competitor AKAP peptide mixture. J, the IC50 (n = 3) of the competitor AKAP peptide mixture for AKAP2 associated with either RSelectAKAP2 (red bar) or WT RII (blue bar).