AKAP9 associates with Epac1 and MTs and is required for the Epac-induced increase in microtubule growth rate. (A) Cosedimentation of AKAP9 and Epac1 with MTs. Cell lysates of 293 cells transfected with flag-tagged construct of AKAP9^2875-3899 fragment and full-length Epac1 were placed on ice to depolymerize the MTs. Samples were incubated with GTP and taxol at 37°C or held on ice (0°C) (negative control). After centrifugation, the pellet sample containing polymerized MTs (P) and supernatants (S) was separated by SDS-PAGE followed by immunoblot analysis. Anti-flag antibody was used to detect Epac1 and AKAP9 (top left panel), and anti-Epac1 antibody was used to specifically identify Epac1 (right panel). Bottom panels, anti–α-tubulin versus β-actin identified the microtubule-enriched compartment (ie, tubulin positive but actin negative). Only the pellet sample at 37°C (P, 37°C) containing polymerized MTs (lacking actin) was enriched in AKAP9 and Epac1 compared with the supernatant (S, 37°C). The lower-molecular-weight anti–flag-band was confirmed to be Epac1, because it was immunoreactive with the anti-Epac1 antibody (right panel). (B) Coimmunoprecipitation of AKAP9 and Epac1. 293T cells were transfected with Epac1-V5 and flag-empty vector alone or the indicated flag-AKAP9 fragments. Lysates (Input) were immunoprecipitated (IP) with IgG control or anti-flag antibody and probed for the presence of AKAP9 and Epac by immunoblotting (IB) with anti-flag (top panel) or anti-Epac1 (bottom panel) antibodies. Epac coimmunoprecipitated only with AKAP9^2875-3899 and AKAP9^1917-2876, and weakly with AKAP9^1229-1917. For panels A and B, 1 of 3 representative experiments is shown. (C) Microtubule regrowth following nocodazole-induced depolymerization. Control (Con) and AKAP9 siRNA (Akp9)–treated HUVECs were incubated with nocodazole on ice and microtubule regrowth was quantified 20 and 30 minutes after nocodazole washout in normal (−) or O-Me-cAMP (O-Me) (+) supplemented medium at 37°C. Cells were stained for α-tubulin and the length of the MTs was determined. *P < .05. n = 3 independent experiments.