ORP10 associates and colocalizes with ORP9 at ER–endosome MCSs. (A) ORP9 as an ORP10-binding protein revealed by MS of ORP10-3xHA immunoprecipitates. The number of ORP9 peptides detected from parental control (Cont) or ORP10-3xHA KI cells is shown. ND, not detected. (B) Parental or ORP10-3×HA KI HeLa cells were lysed and subjected to immunoprecipitation (IP) using anti-HA antibody followed by Western blotting with HA, ORP9, or actin antibody. (C and D) Schematic of ORP10 (C) and ORP9 (D) constructs used in E. Blue, brown, or pink box indicates the PH domain, ORD, or FFAT motif, respectively. (E) GFP, GFP-ORP10 (FL), GFP-ORP10^ΔORD (ΔORD), GFP-ORP10^ΔPH (ΔPH), or GFP-PH^ORP10 (PH) was coexpressed with HA-ORP9 in HEK293T cells, lysed, and subjected to immunoprecipitation using anti-GFP antibody, followed by Western blotting (IB) with antibodies as indicated. (F and G) Cos7 cells expressing GFP-ORP10, mCherry-ORP9, and BFP-VAPA (F) and its quantification for colocalization with ORP10 (G; n = 11 cells). Scale bars, 5 µm (full size) and 1 µm (inset). (H) Western blotting of control or ORP9 KO cells using antibodies indicated. (I and J) Representative images showing endosomal localization of GFP-ORP10 and its association with the ER (mRFP-Sec61β) in control (Cont) or ORP9 KO cells (I) and its quantification (number of ORP10-positive endosomes associated with the ER per cell; J; n ≥ 11 cells). Scale bars, 5 µm (full size) and 1 µm (inset). ****, P < 0.0001.