Stimulus-dependent association of coronin 1 with Gαs.

<p>(A, B) Coronin 1–expressing Mel JuSo cells were left untreated or stimulated with isoproterenol (10 µM, 10 min), lysed, and Gα molecules were immunoprecipitated with the indicated antibodies as described, followed by separation on SDS-PAGE and immunoblotting using anti–coronin 1 (A) or anti-Gα antibodies (B). The appearance of Gα as a doublet may be a result of posttranslational modifications and/or partial cleavage. (C) Coronin 1–expressing Mel JuSo cells were treated with Latrunculin B (4 µM, 30 min; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#pbio-1001820-g005" target="_blank">Figure 5D–F</a>), lysed, and proteins immunoprecipitated using anti-Gα antibodies, followed by SDS-PAGE and immunoblotting. (D) NIE-115 cells were stimulated with isoproterenol (10 µM, 10 min), lysed, and immunoprecipitated using anti–coronin 1 antibodies. Protein complexes were separated by SDS-PAGE and immunoblotted for coronin 1 and the Gα molecules indicated. (E) Coronin 1–expressing Mel JuSo cells or control cells were starved in rolipram (100 µM, 1 h) and stimulated with cholera toxin (1 µg/mL) for 1 h at 37°C prior to stimulation with isoproterenol (10 µM, 4 min). cAMP production was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001820#s4" target="_blank">Materials and Methods</a>.