FIGURE 7.

Acute depletion of COG4 displaces a significant fraction of the Golgi enzymes into CCD vesicles causing their subsequent degradation and glycosylation defects. (A) WB of total cell lysates during time‐dependent depletion of COG4‐mAID shows the expression of Golgi B4GalT1, GalNT2, and MGAT1 enzymes. 10 μg of total cell lysates were loaded and probed with indicated antibodies. β actin was used as a loading control. (B) The graph represents the quantification of A. In the bar graph, values represent the mean ± SD from three independent experiments. Statistical significance was calculated using one‐way ANOVA. ****p ≤ 0.0001, significant. (C, D, E) Airyscan superresolution IF analysis of untreated (control) or IAA treated COG4‐mAID cells stained for (C) GM130 (green) and B4GalT1 (red), (D) GM130 (green) and GalNT2 (red), (E) GM130 (green) and MGAT1 (red), respectively. Scale bars, 20 μm. For better presentation, green and red channels are shown in inverted black and white mode whereas the merged view is shown in RGB mode. (F) Colocalization of Golgi enzymes and GM130 was determined by calculating Pearson's correlation coefficient, >90 cells were analyzed. Statistical significance was calculated by GraphPad Prism 8 using paired t‐test. Here, ***p ≤ 0.001, ****p ≤ 0.0001 (significant) and p ≥ 0.05 nonsignificant (ns). Error bar represents mean ± SD. (G) WB analysis of glycosylation enzymes in Golgi and vesicle fractions. Equal volumes of Golgi (G) and vesicle (V) membrane fractions were analyzed with corresponding antibodies. (H) The graph represents the quantification of vesicle fraction (%) of Golgi enzymes in COG depleted cells compared to control. The Golgi enzyme abundance in vesicles was calculated as a percentage of the immuno signal in the vesicle fractions to the combined signal in Golgi and vesicle fractions from n = 3 independent experiments. Statistical significance was calculated by GraphPad Prism 8 using paired t‐test, ****p ≤ 0.0001, ***p ≤ 0.001, significant. Error bar represents mean ± SD. (I) HPA‐647 lectin blot of total cell lysates obtained during time‐dependent depletion of COG4‐mAID. COG4 KO cells were used as a control. (J) The graph represents the quantification of H. (K) GNL‐647 lectin blot of total cell lysates during time‐dependent depletion of COG4‐mAID. COG4 KO cells were used as a control. (L) The graph represents the quantification of K. Values in bar graphs represent the mean ± SD from three independent experiments. Statistical significance was calculated in GraphPad Prism 8 using one‐way ANOVA. ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05