The VAPs are required to establish/maintain the ERTGoCS. (A) Electron micrograph of a VAP-KD cell and quantification of ERTGoCS in WT and VAP-KD cells. n = 40 Golgi stacks. (B) Quantification of average donor lifetime in donor alone or donor+acceptor VAP-KD and VAP-KO HeLa cells, in cells after treatment with rapamycin (2 min, 200 nM), or after transfection with FLAG-tagged PH-FFAT of OSBP1. The corresponding FRET efficiency is shown on the right. n = 20; data are means ± SD. **, P < 0.01; ***, P < 0.001; Student’s t test. (C–E) Representative immunofluorescence (IF; gray) and FLIM (in color) images showing donor lifetime in VAP-KD and VAP-KO cells (C), in rapamycin-treated cells (D), and in PH-FFAT–expressing cells (E). The pseudocolor scale representing the τ values of TGN46-GFP is the same as in Fig. 2 D. The graphs show the mean τ value distribution curves of TGN46-GFP under the indicated conditions.