ISO induced the translocation of CaMKII-δC and Epac1 to plasma membrane and the phosphorylation of PLB at Thr17 site. (A and B) HEK-293 cells stably expressing WT–β₁-AR were transiently transfected with CaMKII-δC (A) or Flag-Epac1 (B), along with β-arrestin1 (β-Arr1)– (left) or β-arrestin2–YFP (right). Serum-starved cells were stimulated with 10 µM ISO at 37°C. Dual labeling experiments were performed wherein cells were stained for CaMKII-δC and Flag-Epac1 with Texas red, and β-arrestin was visualized by YFP fluorescence. ISO stimulation resulted in marked redistribution of β-arrestin–YFP together with CaMKII-δC (A) or Flag-Epac1 (B) to the membrane (arrowheads). (C) WT, β-arrestin1 KO, and β-arrestin2 KO mice were administered with saline (NS), 5 mg/kg ISO, or 2.5 mg/kg 8-CPT by i.v infusion for 5 min. LV homogenates were immunoblotted with anti–phospho-PLB (p-PLB) at Ser16, anti–phospho-PLB at Thr17, and anti-PLB antibodies. The phosphorylation of PLB at Ser16 (bottom left) and Thr17 (bottom right) was quantified, expressed as fold increase over NS-treated WT mice, and shown as mean ± SEM (n = 5). KO of either β-arrestin1 or -2 inhibited ISO- or 8-CPT–mediated PLB phosphorylation at Thr17. *, P < 0.01 versus NS-treated WT mice; ^#, P < 0.01 versus ISO-treated WT mice; ^‡, P < 0.01 versus 8-CPT–treated WT mice. T-PLB, total PLB. Bars, 10 µm.