Figure 3. Suppression of AC5 by Ric8a in an isoform-specific manner.

HEK-293T cells were transfected with cDNAs of the indicated AC variant in the absence or presence of Ric8a for 72 h. (A) Membrane fractions were collected and assayed for AC activity evoked by forskolin (100 μM). Results are means±S.E.M. for 15 determinations from five independent experiments. (B) Membrane fractions were collected and assayed for AC5 activity evoked by forskolin at the indicated concentrations. The inset is a representative picture which demonstrates the expression levels of AC5 (upper panel) and Ric8a (lower panel) in membrane fractions (100 μg) of HEK-293T cells transfected with the indicated cDNA by Western blot analysis. Results are means±S.E.M. for three determinations (representative of three independent experiments). (C) Cells were harvested and treated either with or without isoprenaline (10 μM) for 20 min at room temperature in the presence of a phosphodiesterase inhibitor [isobutylmethylxanthine (IBMX) 0.5 mM]. Intracellular cAMP levels are expressed as percentages of the cAMP response in the absence of Ric8a in the control, non-treated group. Results are means±S.E.M. for 15 determinations obtained from five independent experiments. Statistical significance was evaluated by one-way ANOVA. *, P<0.01 compared with that in the absence of Ric8a in each group. The cAMP accumulation of the control group obtained from five independent experiments was 659.1±76.0 pmol/10⁶ cells.