ELISA and FRET-based analysis of cAMP production in coronin 1–positive and coronin 1–negative cells in the presence and absence of F-actin.

<p>(A) Coronin 1–expressing or control Mel JuSo cells were left untreated or stimulated with isoproterenol (5 µM, 4 min) and processed for cAMP analysis using ELISA (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#s4" target="_blank">Materials and Methods</a>). The values were normalized to total protein amounts (pmol cAMP/mg protein). The data shown are a representative of three independent experiments. Data represent mean and SEM. (B, C) FRET-based analysis of cAMP production upon stimulation of wild-type or coronin 1–expressing Mel JuSo cells that had been transfected with pICUE3 with isoproterenol (10 µM). (B) the normalized emission ratio (CFP/YFP) time course of coronin 1–expressing (gray) or wild type (red) Mel JuSo cells shown in C (mean, <i>n</i> = 50). (C) Stills from movies representing the normalized CFP/YFP emission ratios. Arrows indicate the time of isoproterenol addition. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s014" target="_blank">Movies S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s015" target="_blank">S2</a>. (D–F) FRET-based analysis of cAMP production upon stimulation of Latrunculin B–treated (4 µM, 30 min) wild-type or coronin 1–expressing Mel JuSo cells (transfected with pICUE3) with isoproterenol (5 µM, at T = 25 s). (D) The normalized emission ratio (CFP/YFP) time course of coronin 1–expressing (grey) or wild-type (red) Mel JuSo cells (mean, <i>n</i> = 30). (E) Coronin 1 and F-actin staining in untreated and Latrunculin B–treated cells. (F) Stills from movies representing the normalized CFP/YFP emission ratios. Arrows indicate the time of isoproterenol addition. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s016" target="_blank">Movies S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio.1001820.s017" target="_blank">S4</a>.