![PI4P-dependent localization of ORP10 at endosome via its PH domain.(A) Confocal images of Cos7 cell expressing GFP-ORP10, RFP-Sec61β, and iRFP-Rab5. Scale bars, 2.5 µm (full size) and 1 µm (inset). (B) Quantification of A showing the percentage of ORP10 colocalized with Rab5 and Sec61β (n = 11 cells). (C) Western blotting of ORP10-3xHA KI HeLa cells transfected with siRNA for control (Cont) or ORP10. (D) Immunofluorescence staining of endogenous ORP10-3xHA with anti-HA antibody in ORP10-3xHA KI cells showing endosome-like punctate structures. Scale bars, 10 µm (full size) and 2 µm (inset). (E) Colocalization of endogenous ORP10 (ORP10-3xHA) with VAPA (AcGFP-VAPA), PI4KIIα (antibody), or Rab7 (GFP-Rab7A) and its quantification (percentage colocalization; n ≥ 10 cells). Scale bar, 1 µm. (F) Schematic of ORP10 constructs (Full [1–766], ΔPH [182–766], PH [77–173], or N-term [1–76]) used in G. Blue or brown box indicates the PH domain or ORD, respectively. (G) Confocal images of Cos7 cells expressing GFP-tagged ORP10 constructs and mScarlet-PI4KIIα. Scale bars, 5 µm (full size) and 1 µm (inset). (H–J) Representative images showing endosomal localization of ORP10 PH domain (2xPHORP10) before (0 min) or after (5 min) recruitment of FKBP fused with Sac1 domain from Sac2/INPP5F (H) or MTM1 (I) by Rab7-FRB and its kinetics (J; mean ± SEM; n = 11 cells for Sac2/INPP5F and MTM1). Scale bars, 5 µm (full size) and 1 µm (inset). Rapa., rapamycin.](https://cdn.rupress.org/rup/content_public/journal/jcb/221/1/10.1083_jcb.202103141/4/jcb_202103141_fig1.png?Expires=1771737604&Signature=kHzuHJzn4c9wjguon5mF2pUBvQ5zS64EfEBAEmhOFOtOrAEynQUm95qxhKzI1nvYeW32sZLqJNDTmMeDN-MaRT5YMiKWp0TW13ky2JjUo0eU4owHznuHis9UNRStIpHa1H6JICee3JHenRKSWADZ3e6PceDeClTh6tVQ8pbjRh6myVW8xirdqVhmCjvG22X8ZQtLXquQ7h5ScWpGBNgKY0MXMcyOpPJc~t1mLsnqjjn3eZYl~xb2eEC~P~3LxXjDiwxag7r0XQpv7Djaj3yhN7rN9xxO3YDW2Eo4KS2OGz1n2fnFLO5o3x2QncEq3r2k2X9mkvGxztxpmCY-TdPCBw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
PI4P-dependent localization of ORP10 at endosome via its PH domain. (A) Confocal images of Cos7 cell expressing GFP-ORP10, RFP-Sec61β, and iRFP-Rab5. Scale bars, 2.5 µm (full size) and 1 µm (inset). (B) Quantification of A showing the percentage of ORP10 colocalized with Rab5 and Sec61β (n = 11 cells). (C) Western blotting of ORP10-3xHA KI HeLa cells transfected with siRNA for control (Cont) or ORP10. (D) Immunofluorescence staining of endogenous ORP10-3xHA with anti-HA antibody in ORP10-3xHA KI cells showing endosome-like punctate structures. Scale bars, 10 µm (full size) and 2 µm (inset). (E) Colocalization of endogenous ORP10 (ORP10-3xHA) with VAPA (AcGFP-VAPA), PI4KIIα (antibody), or Rab7 (GFP-Rab7A) and its quantification (percentage colocalization; n ≥ 10 cells). Scale bar, 1 µm. (F) Schematic of ORP10 constructs (Full [1–766], ΔPH [182–766], PH [77–173], or N-term [1–76]) used in G. Blue or brown box indicates the PH domain or ORD, respectively. (G) Confocal images of Cos7 cells expressing GFP-tagged ORP10 constructs and mScarlet-PI4KIIα. Scale bars, 5 µm (full size) and 1 µm (inset). (H–J) Representative images showing endosomal localization of ORP10 PH domain (2xPHORP10) before (0 min) or after (5 min) recruitment of FKBP fused with Sac1 domain from Sac2/INPP5F (H) or MTM1 (I) by Rab7-FRB and its kinetics (J; mean ± SEM; n = 11 cells for Sac2/INPP5F and MTM1). Scale bars, 5 µm (full size) and 1 µm (inset). Rapa., rapamycin.
