Figure 3

Control and GRASP55 KO clones were transfected with HA‐tagged GSL biosynthetic enzymes, treated with nocodazole (33 µM) for 3 h, and processed for immunofluorescence with anti‐HA (green), anti‐GM130 (red), and anti‐TGN46 (blue) antibodies. The relative position of HA‐tagged enzymes with respect to GM130 and TGN46 was measured by line scanning and expressed as normalized positions of the peak intensity with the start of GM130 peak indicated as Cis and the end of TGN46 peak indicated as trans in the graph. The double‐headed arrows show the distance between the peak localization of GM130 and the enzymes. Scale bar 1 μm.

Control and GRASP55 KO cells were transfected for 16 h with GCS‐HA or LCS‐HA and processed for cryoimmunolabeling with anti‐HA antibody (10‐nm gold particles) and anti‐GM130 antibody (15‐nm gold particles) in case of GCS‐HA and anti‐HA antibody (15‐nm gold particles) and anti‐GM130 antibody (10‐nm gold particles) in case of LCS‐HA. Representative images of the distribution of GCS‐HA and LCS‐HA are shown. Scale bar 200 nm. Red asterisk marks C4 cisterna and blue circles indicate GM130 labeling.

Distribution of indicated enzymes across the Golgi stack was quantified and represented as fraction of Gold particles in each cisternae for GCS‐HA (D) and LCS‐ HA (E) (n indicated in the graph; data are Mean ± SEM).