Colocalization of Sac1 WT or K2A with VAP-A, but not with Bap31, and their proximity to TGN46 or GM130. (A–D) HeLa cells expressing GFP-Sac1 WT or K2A were treated with 2 µg/ml 25-HC for 1 h with (A–C) or without (D) subsequent treatment with 5 µg/ml nocodazole for 2 h. The cells were fixed and visualized with GFP and antibodies against GM130 and VAP-A (A and D), Bap31 (B), or TGN46 (C). Images were subjected to deconvolution processing as described in Materials and methods. Merged images are shown in the second and fourth rows (second row, left panel: panels 1 and 2 [colored in green and red, respectively]; middle panel: panels 1 and 3 [colored in green and red, respectively]; right panel: panels 1, 2, and 3 [colored in green, red, and blue, respectively]; fourth row, left panel: panels 4 and 5 [colored in green and red, respectively]; middle panel: panels 4 and 6 [colored in green and red, respectively]; right panel: panels 4, 5, and 6 [colored in green, red, and blue, respectively]). Arrowheads indicate the small punctate elements containing both GFP-Sac1 and VAP-A. Scale bars, 10 µm. N, nucleus.