ER-localized SCAP interacts through Sac1 with the VAP-A–OSBP complex.(A) Interactions of FLAG-SCAP with Myc-OSBP, Sac1, and VAP-A in HEK 293T cells. Bottom panel shows a schematic representation of the SCAP interactions with VAP, OSBP, and Sac1. (B) Interactions of FLAG-SCAP with Myc-OSBP, Sac1, and VAP-A, but not with HA-CERT or RTN-4B. (C) Interactions of FLAG-Sac1 with VAP-A and Myc-OSBP WT, but not with the PH-FFAT mutant. (D) Interactions of FLAG-SCAP C-term with Myc-OSBP, Sac1, and VAP-A. (E) Establishment of a HeLa stable cell line expressing FLAG-SCAP. Ham, hamster; Hum, human. (F) Interaction of stably expressed FLAG-SCAP with Sac1, but not with RTN-4B or Bap31. VAP-A was detected as a very faint band in the immunoprecipitate (IP) of the stable cell line (lane 4). (G) Interactions of the FLAG-SCAP D451A/L452A mutant with Myc-OSBP, Sac1, and VAP-A. Single and double asterisks denote degraded Myc-OSBP fragments and the Ig heavy chain, respectively. IB, immunoblotted.