Dynabeads

Protein G dynabeads (ThermoFisher Scientific) were coated with 1 μg mouse ST2-Fc (Biolegend), and washed on a DynaMag-2 magnet with PBS 0.02% Tween 20. 100 ng recombinant murine IL-33 (Biolegend) was then mixed with 1 μg HpARI, HpARI_CCP1/2 or HpARI_CCP2/3, and incubated at room temperature for 15 min, prior to adding to ST2-Fc-coated Protein G dynabeads. Beads were washed and bound IL-33 eluted with 50 mM glycine pH2.8, then ran on 4–12% SDS-PAGE gels (ThermoFisher Scientific) under reducing conditions, and transferred to nitrocellulose membranes for western blotting, probing with anti-IL-33 goat polyclonal antibody (R&D Systems AF3626), rabbit anti-goat IgG-HRP secondary antibody (ThermoFisher Scientific) and detected using WesternSure Premium reagent (Licor). Densitometry was carried out using ImageJ, and expressed as fold change from controls at each timepoint.

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).

To further confirm the chemotactic effect of CCL5 on CD8⁺ T cells, naïve CD8⁺ T cells were purified from mouse spleen and activated with Dynabeads containing mouse T-activator CD3 (Biolegend, Cat# 100301)/CD28 (Biolegend, Cat# 102101) and recombinant mouse IL-2 (Biolegend, Cat# 714604). And then, the activated CD8⁺ T cells were seeded in the upper chambers of transwell plates (BD Biosciences) and allowed to migrate for 24 h towards the lower chamber containing medium with different concentrations of CCL5 protein (Pepro Tech, Cat# 250-07).