SCAP is important for PI4P turnover and VAP-A–OSBP complex distribution at ER–Golgi MCSs. (A) Filipin staining in parental HeLa and shSCAP HeLa cells. High magnifications of the boxed areas are shown in the right panels. (B and C) PI4P staining in parental HeLa and shSCAP HeLa cells with (top two rows in C) or without (B and bottom two rows in C) sialyltransferase (ST)–mRFP expression. The graph shows determination of the Golgi PI4P levels in parental HeLa (control) and shSCAP HeLa cells. Data are means ± SEM (n = 139 cells per condition; ****, P < 0.0001; unpaired two-tailed Student’s t test). N, nucleus. (D) BiFC visualization of the OSBP–VAP-A interaction in control (Cont) and SCAP knockdown cells. HeLa cells stably coexpressing Vn-OSBP and Vc-VAP-A were transfected with siRNA. After 72 h, the cells were treated with or without 2 µg/ml 25-HC for 2.5 h. High magnifications of the boxed areas are shown in the insets where brightness/contrast enhancement was applied. The graph shows the percentage of cells with the peripheral BiFC signal of Vn-OSBP–Vc-VAP-A. Data are means ± SEM (n = 3 independent experiments; 100–131 cells per condition; ***, P < 0.005; unpaired two-tailed Student’s t test). (E) Close apposition of CD63- but not of TGN46-positive membranes to the peripheral BiFC signal of Vn-OSBP–Vc-VAP-A in SCAP knockdown cells treated with 2 µg/ml 25-HC for 2.5 h. High magnifications of the boxed areas are shown in the insets. Scale bars, 10 µm (large panels), 5 µm (insets).