Characterizing Arrestin Recruitment to M2R

HEK293 (ATCC) cells lacking endogenous βarr1/2 as described elsewhere^{52 (link),53 (link)} were transiently transfected with a 1:4 DNA ratio of pcDNA-teto-M₂R and GFP-βarr1-WT or the −3xD variant, respectively. Cells were not authenticated or routinely tested for mycoplasma. Cells were split into 35 mm glass bottom microwell dishes (MatTek) and 24 h thereafter serum starved for 2 h in MEM media containing 20 mM HEPES (pH7.4) and 1 mg/mL BSA. Cells were incubated with Alexa-650-labeled FLAG-M1 antibody and NucBlue Live cell stain (Invitrogen) for 1 h at 37 °C, washed two times in starvation media, and imaged using confocal microscopy. In parallel, transfected cells split into 6-well plates were stimulated for 30 min at 37 °C +/− 1 μM iperoxo. Cells were immediately placed on ice and kept at 4 °C for the remainder of the experiment. Cells were washed twice with cold phosphate-buffered saline and then detached with 0.05% EDTA. Cells were resuspended in assay buffer (Hanks balanced salt solution, 20 mM HEPES pH 7.4, 3 mM CaCl₂, 1 mg/mL BSA) and stained with Alexa-650-labeled FLAG-M1 antibody for 30 min at 4 °C. Cells were washed once with assay buffer before analysis by flow cytometry (Bio-Rad S3e Cell Sorter). Data were analyzed using FlowJo software, gating for GFP-positive singlet cells.