Westernsure premium reagent

R&D Systems Duoset kits were used to measure human and murine IL-33 by ELISA, while western blotting was carried out using polyclonal goat anti-mouse IL-33, goat anti-human IL-33 or goat anti-mouse IL-1α (R&D Systems) with a rabbit anti-goat IgG HRP secondary antibody (Thermo Fisher), and detected using WesternSure Premium reagent (Licor).

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.

ELISAs were carried out to manufacturer's instructions for IL-5, IL-13 (Ready-SET-go, ThermoFisher Scientific) and IL-33 (Duoset, Biotechne). IL-33 was also measured in CMT-64 supernatants by western blot – supernatants were ran on 4–12% NuPAGE gels (ThermoFisher Scientific) under reducing conditions, before transferring to nitrocellulose membrane and probing with goat anti-mIL-33 (Biotechne), and rabbit anti-goat IgG HRP secondary antibody (Thermo Fisher), and detected using WesternSure Premium reagent (Licor).