Further characterizing COPII 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: ****P < 0.0001, ns (non-significant) P > 0.05. (A) COPII transport assay assessing the effect of 20 µM of H89 treatment. Quantitation is shown in the below panel, n = 3. Representative confocal images are shown in the upper panel, VSVG (green) and giantin (magenta), bar = 10 µm. (B) Confocal microscopy showing that the Golgi remains intact when cells were treated with 20 µM of H89 for 3 h, n = 6. Representative images examining the distribution of GM130, a Golgi marker, is shown, bar = 10 µm. (C) Confocal microscopy showing the Golgi volume is not changed when cells were treated with 20 µM of H89 for 3 h, n = 6. (D) Confocal microscopy assessing the colocalization of VAP-A with an ER marker (Sec61β) and a Golgi marker (giantin), n = 6. Representative images with scale bars are shown, Sec61 (green), giantin (magenta), VAP-A (blue). (E) Efficacy of siRNA against VAP-A as assessed by western blotting of whole cell lysate, n = 2. β-COP level confirmed similar levels of cell lysates examined. (F) Airyscan confocal microscopy examining the effect of siRNA against VAP-A 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). 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 the 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. (G) Quantitative colocalization results of the above figure is shown, n = 6. (H) 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. (I) Confirmation by confocal microscopy that both longer and shorter NBD-labeled PC is delivered to the Golgi upon their feeding to cells, n = 3. Representative images of NBD-PCs (green) colocalizing with giantin (magenta) are shown on the right, bar = 10 µm. The quantitation of a representative experiment is shown on the left. Source data are available for this figure: SourceData FS1.