FIGURE 3.

Acute COG4 depletion causes relocalization of conserved oligomeric Golgi (COG)-sensitive Golgi SNAREs GS15 and GS28 to the vesicular fraction. (A) Schematic representation of cellular fractionation experiment to prepare Golgi (G) and vesicle (V) fractions from control and IAA treated cells. (B) WB analysis of SNARE proteins (GS15, GS28, STX5, YKT6) in Golgi and vesicle fractions. Equal volumes of Golgi (G) and vesicle (V) membrane fractions were analyzed with antibodies as indicated. (C) The graph represents the quantification of vesicle fraction (%) of SNAREs in COG depleted cells compared to control. SNARE abundance in vesicles was calculated as a percentage of the fluorescent WB signal in the vesicle fraction 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. Here, p ≥ 0.05, nonsignificant, *p ≤ 0.01 (significant). Error bar represents mean ± SD. (D, E) Acute COG4 depletion does not displace the t-SNAREs (STX5) from Golgi but v-SNARE GS15 is relocalizing into vesicles. Airyscan superresolution IF analysis of untreated (control) or IAA treated COG4-mAID cells stained for (D) GM130 (green) and STX5 (red) and (E) GM130 (green) and GS15 (red). 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 SNAREs with GM130 was determined by calculating Pearson’s correlation coefficient and >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