BiFC visualization of SCAP, VAP-A, and Sac1 interactions at ER–Golgi MCSs. (A–F) BiFC visualization of the SCAP–Sac1 or SCAP–VAP-A interaction at ER–Golgi MCSs in HeLa cells coexpressing Myc-OSBP and Vn-fused SCAP in combination with Vc-fused Sac1 (A, C, and E) or VAP-A (B, D, and F). The cells were incubated without (Control [Cont]) or with 2 µg/ml 25-HC for 2.5 h or were cholesterol depleted (Chol Depl) for 3 h. The expression of Vn-SCAP together with Vc-Sac1 (A) or Vc-VAP-A (B) was visualized with an anti-GFP antibody. (C and D) Quantification of perinuclear BiFC signal. Boxes delimit the first and third quartiles and the central line is the median, whereas the cross represents the mean value. The whiskers represent the minimum and maximum values after outlier removal (n = 70–101 cells per condition; *, P < 0.05; ****, P < 0.0001; Kruskal-Wallis multiple sample nonparametric test). N, nucleus. Scale bars, 10 µm.