Fig. 1: Golgin45 is subjected to SUMO1/3 modification.

a Golgin45 is robustly conjugated to SUMO1 and the SUMOylation of Golgin45 is abolished by a SUMO-activating enzyme (SAE) inhibitor TAK981. Hela cells were transiently transfected with the indicated plasmids overnight, then the cells were treated with DMSO or 10 μM TAK981 for 12 h and then SUMO1-modified Golgin45 was analyzed by western blot. b Golgin45 is SUMOylated by both SUMO1 and SUMO3. Hela cells were transfected with myc-SUMO1/3, Flag-UBC9-WT or its dominant-negative (DN) mutation and mCherry-Golgin45 for 18 h, then the cells were lysed and SUMOylation of Golgin45 was examined by western blot. c The SUMOylation sites modified by SUMO1 lie within the N terminal of Golgin45. Hela cells were transfected with mCherry-Golgin45 (121–400) (N terminal deleted truncation) or mCherry-Golgin45 (1–400) (full length) with myc-SUMO1 and Flag-UBC9 for 18 h, the mCherry-Golgin45 SUMOylation was analyzed by western blot. d Golgin45 is SUMOylated at multiple sites. Hela cells were transfected with the indicated plasmids for 18 h. Cells were lysed and analyzed by western blotting using anti-mCherry, anti-Flag and anti-Myc. e Schematic of Golgin45 domains, showing the identified SUMO acceptor sites. TBD Tankyrase binding domain, CC Coiled-coil domain. f–h Mutation of the 8 SUMOylation sites (8-KR) reduces the Golgin45 SUMOylation modified by SUMO1 or SUMO3. HeLa cells were transfected with mCherry-Golgin45 (WT or 8-KR) and Flag-UBC9 (WT or DN) and analyzed by western blot (f). Relative levels of SUMO1 (g) or SUMO3 (h) conjugated mCherry-Golgin45 are presented as mean ± SD. Statistical analysis of relative SUMOylation was performed by one-way ANOVA with Tukey’s multiple comparisons. n = 3 independent experiments. **P < 0.01.****P < 0.0001. i, j Hela cells were transfected with the indicated plasmids for 18 h. Cells were lysed and analyzed by western blotting. Representative blots are shown and experiments were repeated three times.