PITPβ promotes contact between the ER and COPI buds on the Golgi. 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, **P < 0.01, ns (non-significant) P > 0.05. (A) Colocalization of PITPβ with ER marker (Sec61β) and Golgi marker (giantin) as assessed by confocal microscopy using Airyscan, PITPβ (blue), Sec61β (green), giantin (magenta), n = 6. Representative images with scale bars are shown with inset highlighting PITPβ colocalizing with both Sec61β and Giantin (indicated by arrowheads). A reconstruction of this region is also shown, with a line scan along the dotted line providing quantitative information, as well as single-channel images. (B) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a Golgi marker (Giantin). Quantitation of a representative experiment is shown, n = 3. (C) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) and a TGN marker (TGN46). Quantitation of a representative experiment is shown, n = 3. (D) EM tomography showing COPI buds at the Golgi in close proximity to the ER membrane. A representative tomographic image slice is shown in the upper left panel with arrowheads pointing to COPI buds, bar = 200 nm. 3D reconstruction of the Golgi, COPI buds, and ER elements is shown in the lower left panel. Quantitation of a representative experiment is shown on the right, comparing the distance between COPI buds and ER membranes versus the distance between Golgi cisternal margins and ER membranes, n = 3. (E) Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and giantin. Quantitation of a representative experiment is shown on right, n = 3. Representative confocal images are shown on left, PLA signal (red), DAPI (blue). (F) Proximity ligation assay assessing the effect of PITPβ depletion on the colocalization of calnexin and ζ-COP. Quantitation of a representative experiment is shown on right, n = 5. Representative confocal images are shown on left, PLA signal (red), DAPI (blue). (G) Proximity ligation assay examining the effect of siRNA against PITPβ on the proximity between calnexin and TGN46. Quantitation of a representative experiment is shown on right, n = 3. Representative confocal images are shown on left, PLA signal (red), DAPI (blue).