Anti β arrestin 2

SAN tissue from WT and AC_I^–/– mice were flash frozen in liquid nitrogen for Western blotting experiments. The same amount of total protein (5 μg) was loaded in each lane. Membranes were blocked in 3% nonfat dry milk (Bio-Rad) in TBST for 1 hour (room temperature) and then incubated with primary antibodies including anti-HCN4 (1:500 dilution, APC-052, Alomone Labs), anti–β₁-AR (1:1,000 dilution, PA1-049, Thermo Fisher Scientific), anti–β2-AR (1:1,000, PA5-27083, Thermo Fisher Scientific), anti–GRK-5 (1:1,000 dilution, PA5-23189, Thermo Fisher Scientific), anti–β-arrestin-2 (1:1,000 dilution, PA1-732, Thermo Fisher Scientific), and anti-GAPDH (1:5,000, ab8245, Abcam) antibodies, all in 3% nonfat dry milk in TBST overnight at 4°C. On the next day, the membranes were incubated with conjugated secondary antibody (Abcam) for 1 hour at room temperature, and the bands were visualized using Fujifilm LAS-3000 Imager.

Immunoprecipitated proteins were separated by 10% SDS polyacrylamide gel electrophoresis. PVDF membranes were blocked in Tris-buffered saline (5% no-fat milk powder, 0.1% Tween 20), (1 h, RT). Primary antibodies (anti-JNK3, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, #PA5-14421, mAB #2305; anti-PSD95 Clone 6G6 Cayman Chemical, Michigan, USA; anti-JIP1 AbCam Cambridge, UK Ab24449; and anti-β-arrestin2, Thermo Fisher Scientific, Waltham, MA, USA, # PA1-732) were diluted in BSA 5%, 0.1% Tween 20 and incubated overnight at 4 °C. Blots were developed using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) and the ECL chemiluminescence system (Bio-Rad, Hercules, CA, USA).