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. Author manuscript; available in PMC: 2014 Mar 4.
Published in final edited form as: Mol Cell. 2006 Sep 15;23(6):925–931. doi: 10.1016/j.molcel.2006.07.025

Figure 1. AKAP150 Associates with ACV and ACVI.

Figure 1

(A–D) Endogenous AC complexes were isolated from rat brain extract (RBE) by using forskolin-agarose affinity chromatography. Copurifying proteins were identified by RII overlay (A) or Western blots detecting (B) AKAP150, (C) RII, and (D) ACV and ACVI.

(E) Immunoprecipitation of ACs from RBE was performed with antibodies against the ACI, ACII, or ACV/VI isoforms. Immunoblot analysis of each immune complex using anti-AKAP150 (top) and anti-RII (bottom) detected AC-associated proteins.

(F) Measurement of the PKA activity in ACV/VI immune complexes from RBE. Activity measurements were performed with and without the PKA inhibitor PKI. Data are presented as the mean ± SEM from three independent experiments (p < 0.001).

(G) Expression of FLAG-tagged AKAP79 alone or in combination with His-tagged ACV in HEK293 cells was followed by anti-FLAG immuno-precipitation of AKAP79. Coprecipitating AC activity was measured upon stimulation with forskolin and GαS-GTPγS. Data are presented as the mean ± SEM, from three independent experiments, each performed in duplicate (p < 0.001).

(H) Reciprocal immunoprecipitations using the His tag to isolate ACV complexes were immunoblotted with anti-FLAG to detect coprecipitating AKAP79 (top). The expression levels of FLAG-AKAP79 in cell lysates were determined by immunoblot (bottom).