SEP-β2AR relocalizes and internalizes at TfR-pHuji clusters upon stimulation. (A) Portion of a HeLa cell cotransfected with SEP-β2AR and TfR-pHuji before (top) and during (bottom) application of the β2AR agonist isoproterenol (20 µM). (B) Fluorescence in the green and red channels of segmented clusters at pH 7.4 in a representative cell before (a) and during (b) application of isoproterenol. Yellow dots, clusters detected in both channels (238 [a] and 204 clusters [b]); green dots, clusters detected in the SEP channel only (77 [a] and 75 clusters [b]); red dots, clusters detected in the pHuji channel only (127 [a] and 96 clusters [b]). Black dashed lines show linear regressions of all TfR-pHuji clusters (R = 0.41 [a] and R = 0.87 [b]). Green and red lines show the lower limits of SEP and pHuji cluster detection, respectively. (C) Proportion for n = 7 cells of TfR-pHuji clusters enriched in SEP-β2AR (yellow) or not (red) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and green), before (a) and after (b) application of isoproterenol. (D, a) Frequency of events represented as cumulative number in seven cells of events detected with TfR-pHuji (left) and SEP-β2AR (right) during the course of the experiment, normalized at the end (17 min). Dotted lines are regression lines of the data before agonist application, extrapolated to the entire recording. (b) The frequency of scission events detected with SEP-β2AR (green) is significantly higher during isoproterenol application as compared with baseline (*, P < 0.01), whereas the frequency of TfR-pHuji–detected scission events (red) remains unchanged. Error bars represent SEM.