FIGURE 4.

The rapid COG4 depletion has no effect on the localization of coiled-coil tether p115 but displaces giantin, golgin84, and TMF1 from Golgi. Airyscan superresolution IF analysis of untreated (control) or IAA treated COG4-mAID cells stained for (A) GM130 (green) and golgin84 (red), (B) GM130 (green) and p115 (red), and (C) GM130 (green) and giantin (red). Scale bars, 20 μm. For the better presentation, green and red channels are shown in inverted black and white mode whereas the merged view is shown in RGB mode. (D) Colocalization of tested golgins with GM130 was determined by using Pearson’s correlation coefficient, >90 cells were analyzed. Statistical significance was calculated by GraphPad Prism 8 using paired t-test. Here, p ≥ 0.05, nonsignificant (ns), ****p ≤ 0.0001 (significant). Error bar represents mean ± SD. (E) WB analysis of tethers (p115, golgin84, TMF1) in Golgi and vesicle fractions. Equal volumes of Golgi (G) and vesicle (V) membrane fractions were analyzed with corresponding antibodies. (F) The graph represents the quantification of vesicle fraction (%) of golgins in COG-depleted cells compared to control. The abundance of p115, golgin84, and TMF1 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 ≤ 0.05, significant, p ≥ 0.05, nonsignificant (ns). Error bar represents mean ± SD