HRS is required for activated EGFR recycling. (A) HeLa cells were treated with the indicated siRNA over 120 h and serum-starved for 2 h before stimulation with 1 ng/ml EGF over the indicated time course and lysis in RIPA buffer. IB, immunoblot. Molecular masses are given in kilodaltons. (B) Quantification of degradation normalized to time 0 min. (C and D) Quantification of relative fluorescence intensity of EGFR in EEA1-positive endosomes. Values were normalized to endosome size. (C) EGFR endosomal intensity after 30-min treatment with 1 ng/ml EGF. (D) EGFR intensity in EEA1-positive endosomes over time course of stimulation with 1 ng/ml EGF, values were normalized to time point 0 for each condition. n = 3; error bars indicate SEM; approximately >75 cells total. (E–G) HeLa cells treated as before, followed by fixation in 4% PFA/PBS and staining with antibodies targeting EEA1 and EGFR. Sum intensity projection images. ***, P < 0.001; ****, P < 0.0001. Statistical analysis, one-way ANOVA with Dunnett’s post hoc test. Bars: (main images) 10 µm; (insets) 2 µm. MRG, merge.