HRS is required for receptor recycling. (A) HeLa cells were treated with siRNA for 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting EEA1 and EGFR. (B) Pearson’s R correlation value between EGFR and EEA1. Single confocal slice images. (C) MDA–MB-231 cells were depleted for 120 h with siRNA before fixation in 4% PFA/PBS and labeling with antibodies targeting EEA1 and EGFR. Single-slice images. Bars: (main images) 10 µm; (insets) 2 µm. MRG, merge. (D) Pearson’s R correlation value between EGFR and EEA1. (B–D) n = 3; all data points plotted; >10 images per condition; >150 cells total. (E) Cell surface levels of EGFR were measured by flow cytometry. The mean fluorescent intensity was plotted as a percentage of the siNT control for each individual experiment (n = 4). (F) Total EGFR levels in cell lysates normalized to siNT (n = 3). (G) siHRS- and control-depleted HeLa cells were transfected with EGFR-paGFP fusion constructs and mCherry-RAB4. Trafficking from the endosome was measured by quantifying the decrease in GFP fluorescence in the endosome normalized to rate of photobleaching (n = 9 over two independent experiments). (H) MDA–MB-231 cells were treated with the indicated siRNA for 120 h before being surface-labeled with N-hydroxysuccinimide–SS-biotin on ice and subsequently warmed to generate an internal pool. Then, surface biotin was stripped. The cells were warmed for 7.5 min or 15 min to allow for recycling. The cell surface was stripped again to determine the percentage of recycled receptor compared with total internal pool (n = 3). Error bars indicate SEM. **, P < 0.01; ***, P < 0.001. Statistical analysis, one-way ANOVA with Dunnett’s post hoc test, except B and H are t tests.