Further characterizing how PITPβ promotes COPI transport and contact formation. 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: ns (non-significant) P > 0.05. (A) Efficacy of siRNA against PITPα, as assessed by western blotting of whole cell lysate, n = 2. β-COP level confirmed similar levels of lysate examined. (B) COPI transport assay assessing the effect of siRNA against PITPα. The quantitation of a representative experiment is shown on right, n = 3. Representative confocal images are shown on the left, VSVG-KDELR (green), giantin (magenta), bar = 10 µm. (C) Reduction of PC levels in Golgi membrane upon PITPβ depletion as assessed by untargeted lipidomics, n = 3. Results are shown as a heatmap. PC species are categorized based on their total acyl chain length and unsaturations. (D) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) with a Golgi marker (giantin). Representative images with scale bars are shown, Sec61 (green), and giantin (magenta). The inset of each image is shown with dotted lines specifying the different planes of visualizing the image. The position of an image visualized in the XZ plane is highlighted by the blue dotted line, and its line scan is shown in the upper right corner of each condition. The position of an image visualized in the XY plane is highlighted by an orange dotted line, and its line scan is shown in the lower left corner of each condition. Arrowheads in line scans highlight areas of colocalization. (E) Airyscan confocal microscopy examining the effect of siRNA against PITPβ on the colocalization of an ER marker (Sec61β) with a TGN marker (TGN46). Representative images with scale bars are shown, Sec61 (green) and TGN46 (magenta). The inset of each image is shown with dotted lines specifying the different planes of visualizing the image. The position of an image visualized in the XZ plane is highlighted by a blue dotted line, and its line scan is shown in the upper right corner of each condition. The position of an image visualized in the XY plane is highlighted by orange dotted line, and its line scan is shown in the lower left corner of each condition. Arrowheads in line scans highlight areas of colocalization. (F and G) Comparing the colocalization between original versus 90° rotated images to confirm that the detected colocalizations are not random. Quantitation of a representative experiment is shown, n = 2. Source data are available for this figure: SourceData FS2.