Figure 2. Intact β2-AR trafficking into and out of repositioned endosomes.

A-D. HEK293 cells were pre-treated with ethanol (“Vehicle”) or 1μM rapamycin (“+Rapa”) for 30 min. Cells were fixed and imaged by immunofluorescence microscopy. A. HEK293 cells co-expressing the CID machinery and Flag-β2-AR visualized by immunofluorescence microscopy. β2-AR (red) is present at the cell surface in the absence of β2-AR stimulation. B. β2-AR colocalizes with early endosomes (green) after 20 min of activation with 1μM isoproterenol (“Iso”). β2-AR-containing endosomes selectively redistribute toward the cell periphery upon rapamycin application. Scale bar = 10μm. White arrow indicates repositioned endosomes. C. Endosome redistribution with rapamycin does not impact β2-AR internalization or recycling compared to vehicle-treated cells. Internalization was induced as described in B. Recycling was induced following internalization by application of 10μM alprenolol for 1 h (see also Supplementary Fig. 3). Object-based colocalization analysis was used to calculate internalized or recycled β2-ARs as the ratio of [number of β2-AR puncta colocalized with EEA1]/[total β2-AR puncta] (see “Methods” for details). Data are mean from n = 3 biologically independent replicates ± s.e.m.; 33 cells total/condition; **** = p < 0.0001 by two-way ANOVA test with Tukey.