(a) Total endogenous AC2 and AC5/6 in HeLa cells were examined following overexpression of HA-XIAP and its H467A and F495A mutants and TRIP-Br1-FLAG (quantification in Figure 7—figure supplement 1). (b–c,) Mutation to Arg of K1065 in HA-AC2 and K1247 in HA-AC5 abolished XIAP/TRIP-Br1-induced ubiquitination and degradation of HA-AC2 (b) and HA-AC5 (c), respectively, in HEK293T cells. The experiments shown here are representative of 3 independent experiments. (d–e) Knocking out TRIP-Br1 or the RING domain of XIAP in mice increased the expression of AC2 and AC5/6 in heart tissue. Transferrin receptor or Na/K ATPase: membrane-protein negative control; actin: loading control. *, different for wild-type (WT), p≤0.036, n = 3; **p≤0.006, n = 8. (f), Net cAMP production in response to IBMX (ΔIBMX, 100 μM) or forskolin (ΔFSK, 10 μM) in heart tissues of wild-type (WT), TRIP-Br1 knockout, and XIAP ΔRING mice. ΔIBMX and ΔFSK are the differences between IBMX-stimulated and control cAMP production, and between forskolin/IBMX- and IBMX-stimulated cAMP production, respectively (inset). *, different from WT, p≤0.04; **p≤0.006; ***p≤0.001, n = 8 wk-old females for all three groups. (g) Basal heart rates of wild-type (WT), TRIP-Br1 knockout, and XIAP ΔRING mice. **, different from WT, p=0.0049; *, p=0.032; 8-wk-old male and female littermates of WT (n = 5) and TRI-BR1 knockout mice (n = 6) or 4-wk-old male littermates of WT (n = 5) and XIAP ΔRING mice (n = 6) were used for experiments. The mice were anesthetized with Avertin. The difference in the basal heart rates of 4-wk-old and 8-wk-old WT mice has been reported before (Moghtadaei et al., 2016).