Fig. 2: Terminal glycosylation is impaired by Sac1 depletion.

a Top terms in gene ontology enrichment analysis of the downregulated proteins in Sac1-degron A431 cells treated with IAA for 3 h compared to DMSO control (-Log₁₀ False Discovery Rate (FDR) with a fold change threshold of ≥ 1.30 or ≤ 0.77 and an adjusted p-value of ≤ 0.05). b Schematic overview illustrating the changes in protein levels of glycosylation enzymes in Sac1-degron A431 cells after 3 h IAA treatment. The Log₂ fold change (Log₂FC) of individual enzyme is presented using a color gradient from magenta (−2) to green ( + 2). Glycosylation starts in the ER, where precursor glycan chains are added to the protein. The glycans are modified sequentially by glycosidases and glycosyltransferases along the ER and Golgi stacks. c Western blot analysis of TGN46 and B4GALT1 in Sac1-degron A431 cells treated with IAA for 0 to 4 h. d Bar graph showing normalized intensity of B4GALT1 and TGN46 (mean ±S.E.M) quantified from (c). n = 3 independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test. TGN46: n.s. p = 0.9964, p = 0.5307, *** p = 0.0004, p = 0.0001. B4GALT1: n.s. p = 0.8336, p = 0.9872, p = 0.2245, ** p = 0.0006. e Western blot analysis of lysates of HEK293A Sac1-degron cells transfected with pcDNA3.1 (empty vector), EGFP-Sac1-WT (Sac1^wt) or EGFP-Sac1-C389S (Sac1^mut), treated for 6 h with DMSO or IAA and blotted for B4GALT1 and GFP (Sac1). f Graph depicts the B4GALT1 degradation efficiency (mean ±S.E.M) in Sac1-degron HEK293A cells treated for 6 h with IAA and rescued with Sac1^wt or Sac1^mut. n = 5 independent experiments. One-way ANOVA with Tukey’s multiple comparisons test. n.s. p = 0.6379, ****p < 0.0001.