Anti β2 ar

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.

HEK293T cells were plated onto glass coverslips and transfected with plasmids encoding FLAG-tagged 6TM-MOR alone or with pOPRSVI/MCS-β₂-AR as described above. 48 h after transfection, cells were fixed in -20^°C methanol for 10 min, permeabilized with 0.25% Triton X-100 and blocked with 5% BSA for 40 min followed by immunostaining (primary antibody, overnight at 4 °C). Primary anti-FLAG (DYKDDDDK) antibody (Sigma-Aldrich) was used at a 1:1,000 dilution, anti-Calnexin (Abcam) and anti-Na/K ATPase α1 (Santa Cruz Biotechnology) antibodies were diluted 1:300, and anti-β₂-AR 1:500 (Thermo Fisher Scientific). After washing with TBS, coverslips were incubated with appropriate secondary antibodies conjugated to Alexa Fluor 488 or 594 (Life Technologies) together with Hoechst 33342 (Sigma-Aldrich) for 1 h at room temperature. Samples were imaged using a Zeiss LSM 710 confocal microscope and images were collected and analyzed with ZEN Black software (Carl Zeiss).