Western blot analysis

Adult cardiomyocytes isolated from NC and HFD mice were stimulated with ISO (100 nmol l⁻¹) after incubation with or without pertussis toxin (1 mg ml⁻¹ for 3 h). Left ventricular extracts or adult cardiomyocyte lysates were homogenized at 4°C for Western blotting using standard methods previously described (Fu et al. 2014). Membranes were probed with antibodies directed at β₁AR (V‐19, 1:500 dilution), β₂AR (M‐20, 1:500 dilution), GRK2 (C‐15, 1:1000 dilution) and Gα_i (C‐10, 1:1000 dilution) from SCBT (Santa Cruz, CA, USA), phospho‐troponin I (Ser23/24, #4004, 1:2000 dilution) and troponin I (#4002, 1:2000 dilution) from Cell Signaling (Danvers, MA, USA), SERCA (2A7‐A1, 1:1000 dilution; Thermo, Rockford, IL, USA), phospho‐phospholamban (Ser16, 1:5000 dilution; Bradilla, London, UK), phospholamban (MA3‐922, 1:1000 dilution; Affinity Bioreagent, Golden, CO, USA) and γ‐tubulin (T6557, 1:5000 dilution; Sigma‐Aldrich, St Louis, MO, USA). The primary antibodies were revealed with fluorescent‐conjugated secondary antibodies using an Odyssey scanner (Li‐COR Biosciences, Lincoln, NE, USA). The signal was quantified by Image Studio software version 2.1 (Li‐COR Biosciences).