Figure 4: The abundance of several Golgi enzymes is reduced in GS28 KO cells but to a lesser degree than in COG depleted cells. Enzyme reduction in SNARE-depleted cells did not significantly alter glycosylation of lysosomal and Golgi glycoproteins.

(A) WB analysis of Golgi glycosylation enzymes in WT, GS28 KO, GS15 KO and COG4 KO cells. While the total amount of B4GALT1, MGAT1, GALNT3 and GALNT2 is not as severely depleted in GS28 KOs compared to COG4 KOs, their levels are significantly reduced compared to the WT. (B) Quantification of fold changes in enzyme levels with respect to WT from biological triplicates. (C) WB showing electrophoretic mobility and abundance of glycoproteins; (D) Quantification of the fold change in glycoprotein levels from biological triplicates with respect to WT. (E) Lectin blot detects the total amount of intermediate O-glycan - Tn antigen labeled by HPA-647, and N-glycan – high mannose labeled by GNL-647, in cell lysates. (F) Quantification in the fold change in HPA and GNL staining intensity with respect to WT shows a significant increase in the accumulation of immature glycoproteins in GS28 KOs, but this is not as dramatic as in COG4 KOs. **p<0.001, ***p<0.0001