HRS is required for the endosomal recruitment of WASH. (A) HeLa cells were treated with the indicated siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting HRS and WASH. Maximum-projection images. (B) Airyscan single confocal slice images of HeLa cells fixed and labeled for WASH and HRS. (C and D) PLA of HeLa cells probed for both HRS and WASH or technical single WASH or HRS antibody controls. Data represented as mean number of signals per cell. Maximum-projection images (nuclei stained with DAPI). Bars, 20 µm. (E and F) HeLa S3 Flp-In cells stably transfected with GFP-HRS (mouse) were treated with siRNA targeting endogenous HRS over 120 h before fixation in 4% PFA/PBS or lysis in NP-40 buffer. Single confocal slice images. Molecular masses are given in kilodaltons. IB, immunoblot. (G) Quantification of Pearson’s R correlation between EEA1 and WASH (>150 cells total). (H and I) HeLa cells were treated with siRNA over 120 h before fixation in 4% PFA/PBS and labeling with antibodies targeting EEA1 and 647-phalloidin. Quantification of sum intensity of actin on endosome. Maximum-projection images (>30 cells total). n = 3; error bars indicate SEM. **, P < 0.01; ***, P < 0.001. Statistical analysis, one-way ANOVA, Dunnett’s post hoc test. Bars: (main images) 10 µm; (insets) 2 µm. MRG, merge.