Ab175474

Cells were cultured in 35‐mm ibidi dishes. Once 70% confluency was reached, the media was extracted and 200 μl of ice‐cold 4% paraformaldehyde (PFA) was incubated for 10 mins. Cells were washed three times with cold PBST and blocked using 1% BSA for 30 mins. Primary antibodies directed against β₂AR (Abcam; ab182136), Gαs (Novus; NBP1‐49874), or βarr2 (Mybiosource; MB52522670) at a dilution of 1:300 for 1 h at room temperature. Cells were washed three times with PBST and incubated with the secondary antibodies anti‐rabbit Alexa 488 (Abcam; ab150077), anti‐goat Alexa 568 (Abcam; ab175474) at a concentration of 1:1000, or with anti‐rabbit Alexa 594 (Jackson Immunoresearch; 111‐585‐003) at a 1:200 dilution. Because two secondary antibodies had reactivity to the same species, the primary and secondary antibody incubation for each targeted protein was done separately with an extra blocking step in between to avoid cross‐reactivity of the secondary antibodies. The secondary antibodies were incubated for 1 h at room temperature in a humidified chamber and washed three times with PBST. Finally, cells were mounted using fluoro‐gel II with DAPI (electron microscopy services) and imaged using a DMi8 confocal laser scanning microscope (Leica).