Identification of SCAP as a novel component of Sac1-positive ER–Golgi MCSs. (A) Juxtanuclear localization of the GFP-Sac1 WT and K2A mutant in HeLa cells. (B) Colocalization of GFP-Sac1 WT or K2A with VAP-A and their proximity to TGN46. HeLa cells expressing GFP-Sac1 WT or K2A were treated with 2 µg/ml 25-HC for 1 h. Images were subjected to deconvolution processing as described in Materials and methods. The graphs on the right show the fluorescence intensity of GFP-Sac1 WT or K2A (green), and VAP-A or TGN46 (magenta) along the respective white lines shown in the merged images. N, nucleus. (C) iFRAP in HeLa cells expressing GFP-Sac1 WT, K2A, or NA-GFP. The areas delimited by a red line were bleached as described in Materials and methods. The graph shows quantification of the fluorescence intensity of the indicated proteins in the nonbleached, juxtanuclear region. Data are means ± SEM (n = 4 cells per condition; ****, P < 0.0001; one-way ANOVA multiple comparison test). (D) Silver staining of immunoprecipitated proteins with FLAG-Sac1 WT, FLAG-Sac1 K2A, or FLAG in HEK 293T cells. The arrow indicates FLAG-Sac1. Asterisks denote protein bands containing COPI components. SCAP was identified in the protein band boxed with a red line. (E) Peptides of VAP-A, VAP-B, OSBP, and SCAP, which were identified by MS analysis of FLAG-Sac1 K2A immunoprecipitates (lane 2 in D). (F) Interaction of endogenous Sac1 with SCAP, but not with calnexin or RTN-4B. HEK 293T cell lysate was incubated with Dynabeads Protein G coupled with control (Cont) IgG or an anti-Sac1 antibody, and the cell lysate (Input) and immunoprecipitates (IPs) were immunoblotted (IB) with the indicated antibodies. Asterisk denotes nonspecific bands. Scale bars, 10 µm. AU, arbitrary units.