COPI vesicle reconstitution confirming PITPβ acts in vesicle fission. Quantitative data are shown as mean ± SD, with the number of independent experiments indicated. Statistics was performed using the two-tailed Student’s t test: ****P < 0.0001, ***P < 0.001, ns (non-significant) P > 0.05. (A) Western blotting assessing the release of PITPβ and BARS from Golgi membrane upon salt wash using 3 M KCl, n = 4. (B) Western blotting assessing the depletion of PITPβ from Golgi membrane by treating cells with shRNA against PITPβ, n = 4. (C) The COPI vesicle reconstitution system was performed. The left panel shows the quantitation of vesicle formation, n = 5. The right panels show representative EM images of bud accumulation on the Golgi membrane that had been depleted of PITPβ. Arrows highlight the constricted neck of budding vesicles, bar = 50 nm. (D) Depletion of PITPβ from Golgi membrane that inhibits COPI vesicle formation is rescued when ER-like liposomes that contain sPC and VAP-A are added along with wild-type PITPβ, but not when mutant forms that cannot interact with coatomer (KK mut) or is defective in catalytic activity (C94A) are added, n = 4. (E) Schematic summarizing the role of PITPβ in COPI vesicle fission. Source data are available for this figure: SourceData F7.