Fig. 4: VdUlpB regulates VdEno SUMOylation.
a VdEno is a SUMOylation substrate and deSUMOylated by VdUlpB. Co-incubation of Strep-Tagged VdEno and VdSUMO in the presence of E1, E2, and ATP at 37 °C for 2 h. The products were treated with VdUlpBCD or VdUlpBCDm at room temperature for 4 h followed by Western blotting with anti-Strep or anti-human SUMO1 antibody. b DeSUMOylation of VdEno by VdUlpB in vivo. The Strep-VdSUMO or VdEno-Flag constructs were expressed in WT V592, or co-expressed in the V592 and VdΔulpb mutant. Total protein from these strains was immunoprecipitated with anti-Flag beads and immunoblotted with anti-Flag or anti-Strep antibody. Ponceau staining was used as a loading control. c Conformation of VdEno SUMOylation residues in vitro. In the presence of E1, E2, and ATP, Strep-Tagged VdEno or VdEno5K/5R was co-incubated with VdSUMO at 37 °C for 2 h followed by western blotting with anti-Strep or anti-human SUMO1 antibody. d Conformation of VdEno SUMOylation residues in vivo. VdEno-Flag or VdEno5K/5R -Flag constructs were co-expressed with Strep-VdSUMO in the VdΔulpb mutant. Total protein from these samples was immunoprecipitated with anti-Flag beads and immunoblotted with anti-Flag or anti-Strep antibody. Ponceau staining was used as a loading control. The experiments in a–d were repeated independently three times with similar results. Source data are provided as a Source Data file.
