BiFC visualization of VAP-A, OSBP, and Sac1 interactions at ER–Golgi MCSs. (A and B) No BiFC signal in HeLa cells with single expression of Vn-fused proteins (A) or Vc-fused proteins (B). (C and D) BiFC visualization of OSBP–VAP-A (C) and OSBP–Sac1 (D) interactions at ER–Golgi MCSs. The coexpression of Vn-OSBP WT or FF/AA with Vc-VAP-A (C) or Vc-Sac1 (D) was visualized with an anti-GFP antibody. The BiFC signal was enhanced by treatment of cells with 2 µg/ml 25-HC for 2.5 h. Vn-OSBP FF/AA showed a reduced BiFC signal compared with WT. Scale bars, 10 µm.