Figure EV5. OSBP1 mediates PtdIns(4)P relocation to the ER to control SL homeostasis at the TGN .

1. Cells transiently expressing mCherry‐OSBP1 wt or the mCherry‐OSBP1‐FF>AA mutant were fixed and permeabilized as in Fig 3 and stained with anti‐PtdIns(4)P (green). The dashed line indicates mCherry‐OSBP1‐transfected cells. Scale bar, 10 μm.
2. Cells KD for OSBP1 were treated with myriocin (2.5 μM) for 24 h and fed with increasing concentrations of D‐Sph for 2 h. ³H‐D‐Sph (≈5 nM) was mixed as a tracer with its non‐radioactive counterpart. The percentage of total radioactivity associated with SM (left panel), GlcCer (middle panel) and Cer (right panel) in mock (grey) and OSBP1‐KD cells (red) was quantitated after lipid extraction and HPTLC separation. The blue dashed line represents the values obtained under BFA treatment (from Fig 1B). Data are means ± SEM from at least 4 independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001; according to two‐tailed Student's t‐test.