Localization of ORP10 at endosomes and the ER–endosome MCSs. (A and B) Representative images of Cos7 cells expressing GFP-ORP10 and mScarlet-PI4KIIα (A) or RFP-Sec61β (B). Scale bars, 5 µm (full size) and 1 µm (inset). (C) Colocalization of endogenous ORP10 (ORP10-3xHA) with Rab5 (GFP-Rab5) or Sac2 (GFP-INPP5F-D460A) and its quantification (percentage colocalization; n ≥ 10 cells). Scale bar, 1 µm. (D and E) Representative images showing endosomal localization of OSBP PH domain (OSBP-PH) before (0 min) or after (5 min) addition of rapamycin (Rapa.) to recruit FKBP fused with Sac1 domain from Sac2/INPP5F by Rab7-FRB (D) and its kinetics (E; mean ± SEM; n = 9 cells). Scale bars, 5 µm (full size) and 1 µm (inset). (F and G) Representative images showing endosomal localization of HRS Fyve domain (2xFYVE) before (0 min) or after (5 min) addition of rapamycin to recruit FKBP fused with MTM1 by Rab7-FRB (F) and its kinetics (G; mean ± SEM; n = 11 cells). Scale bars, 5 µm (full size) and 1 µm (inset). (H and I) Representative images showing endosomal localization of ORP10 before (0 min) or after (5 min) recruitment of FKBP fused with Sac1 domain from Sac2/INPP5F by FRB-Rab7 (H) and its kinetics (I; mean ± SEM; n = 7 cells). Scale bars, 5 µm (full size) and 1 µm (inset). (J) Western blotting of cells transfected with siRNA for control (siCont), VAPA (siVAPA), or VAPB (siVAPB) by antibodies indicated. (K and L) Localization of AcGFP-ORP10 in control (Cont) or VAPA/VAPB double-KD (DKD) cells (K) and its quantification (the number of ORP10-positive endosomal structures associated with the ER). In L, n = 11 cells for control, n = 11 cells for VAPA/B DKD. Scale bars, 5 µm (full size) and 1 µm (inset). ****, P < 0.0001.