EHD1 and other regulators for retrograde membrane trafficking at endosomes in ORP10 KO cells. (A) Confocal microscopic images of control (Cont) or ORP10 KO cells showing localization of endogenous EHD1 and SNX1. Scale bars, 5 µm (full size) and 1 µm (inset). (B) Quantification of EHD1 fluorescence (Fluo.) at SNX1-positive endosomes shown in A (mean ± SEM; n = 40 cells). (C–F) Representative immunofluorescence images of endogenous EEA1 and F-actin (C), VPS35 (D), or SNX1 (E), and endogenous SNX1 and transiently expressed GFP-FAM21 (F) in control (Cont) or ORP10 KO HeLa cells. Scale bars, 5 µm (full size) and 1 µm (inset). (G) Western blotting of cells transfected with siRNA for control (siCont) or EHD1 (siEHD1-1) by antibodies as indicated. (H) Quantification of times required for completion of tubule fission in cells transfected with siRNA for control (siCont) or EHD1 (siEHD1-1; mean ± SEM; n = 35 fission events from 11 cells for control, n = 26 fission events from 13 cells for EHD1 KD). ****, P < 0.0001.