Actin binding can overcome Ub sorting at the endosome. (A) Schematic of TrfR chimeras. (B) HeLa cells were transfected with indicated constructs and then incubated in 50 µg/ml FITC-Trf for 15 min. The cells were washed in PBS and incubated in fresh medium with unlabeled Trf to chase for 1 h before fixation and staining. MRG, merge. (C) Pearson’s R values between the receptor (myc) and FITC-TrfR were calculated using Slidebook software (10 images approximately >150 cells total). n = 3. Error bars indicate SD. Bars: (main images) 10 µm; (insets) 2 µm. (D) HeLa S3 cells were transfected with the TrfR chimeras and treated as before except the recycling was stopped at 0, 15, and 30 min, and the retained FITC-Trf was measured by flow cytometry (n = 5). (E and F) HeLa cells were transfected with indicated fusion constructs for 24 h before treatment with CHX to block protein synthesis for the indicated time course. n = 3. Error bars indicate SEM. All experiments were performed in the presence of 50 µg/ml leupeptin and 100 µg/ml CHX except E and F, where leupeptin was omitted. *, P < 0.05; ***, P < 0.001. Statistical analysis, one-way ANOVA for all comparisons, with Dunnett’s post hoc test. Images taken from a single slice.