Anti myc 9e10

Purified Sgo1 (1 µM) was mixed with 5 mM ATP, 15 µM SUMO, 0.5 µM E1 (unless otherwise indicated), 0.5 µM E2 (unless otherwise indicated), and 0.1 µM (unless otherwise indicated) Siz1 (167–508) or Siz2 in a reaction buffer consisting of 25 mM Hepes, pH 7.5, 150 mM KCl, 10 mM MgCl₂, 15% glycerol, 0.1% NP-40, 0.1 mM DTT, and 0.25 mM PMSF. The reaction was incubated at 30°C for 2 h (unless otherwise indicated). For detection of SUMOylated Sgo1, the reaction was boiled in SDS sample buffer before analysis by anti-V5 Western blotting.
For Rts1 binding assay, the product of in vitro SUMOylation was incubated with anti-V5 (#MCA1360; Bio-Rad AbD Serotec)–coupled protein G magnetic Dynabeads (Thermo Fisher Scientific) in binding buffer A (25 mM Hepes, pH 7.5, 150 mM KCl, 2 mM MgCl₂, 15% glycerol, 0.1% NP-40, 0.1 mM EDTA, 0.5 mM EGTA, and 0.25 mM PMSF) for 2.5 h at 4°C. After washing three times with binding buffer A, beads were incubated with extract from strain AMy8832 for 2 h at 4°C. Beads were washed five times with binding buffer A + 10 mM N-ethylmaleimide and were heated at 65°C for 15 min to elute bound proteins. Sgo1 binding assay was performed similarly, except that Rts1-9Myc was immunoprecipitated from lysate of strain AM8832 by anti-Myc (9E10, #626802; BioLegend)–coupled protein G Dynabeads and subsequently incubated with products of in vitro SUMOylation (with or without ATP).