Imaging intra-Golgi secretory transport by side-averaging. (A–D) The intra-Golgi transport of VSVG by side-averaging. HeLa cells transiently coexpressing GalT-mCherry and the RUSH reporter, VSVG-SBP-GFP, were first incubated with nocodazole to induce the formation of ministacks. Cells were subsequently treated with biotin and CHX in the presence of nocodazole to chase VSVG-SBP-GFP along the secretory pathway. Cells were fixed and immunostained for endogenous GM130 to acquire the LQ of VSVG-SBP-GFP (A). In the LQ-vs.-time plot, the data points are colored blue. Error bar, mean ± SEM. n, the number of ministacks analyzed. Data were fitted to the first-order exponential decay function (red line). The plateau and adjusted R² (adj. R²) are from the fitting. Alternatively, fixed cells were immunostained for endogenous giantin to side-average VSVG-SBP-GFP. In B, LQ^side values were acquired from side-average images and are colored blue. Similar to A, LQ^side-vs.-time plot was fitted to the first-order exponential decay function. In C, the panel is organized as in Fig. 2, A–D. The exception is the second row, in which axial line intensity plots are presented with the x and y axis swapped. In D, the normalized radius against time plot shows the change of the radius of VSVG-SBP-GFP during its intra-Golgi transport. Normalized radii were calculated using the radial mean intensity profile of the corresponding en face average images. (E–J) The intra-Golgi transport of SBP-GFP-CD8a-furin WT (E–G) and Y + AC mutant (H–J) by side-averaging. The experiments and the organization of panels are similar to those in A–C. Horizontal green lines in G and J represent the axial position of CLCB. In C, G, and J, scale bars, 200 nm.