*4.10. Pull-Down Assay*

Pull-down assay was performed using Glutathione Sepharose 4B resin (GE Healthcare, Chicago, IL, USA). First, 4 μM of GST-IL-33 was added to the resin and incubated for 30 min at 4 ◦C with gentle shaking. The resin was washed 4 times with wash buffer C (50 mM Tris-HCl pH = 7.5 and 150 mM NaCl) and incubated for 30 min at 4 ◦C with gentle shaking upon the addition of 4 μM of ST2-Fc. Subsequently, C2\_2E12 at a series of concentrations (0.4, 4, 40, and 400 μM) was added to the resulting resin with further incubation for 30 min at 4 ◦C. After 4 times of washing, proteins were separated by SDS-PAGE on a 12% gel, stained with Coomassie Brilliant Blue, or transferred onto a PVDF membrane (45 mA for 60 min). Protein bands on the PVDF membrane were visualized by immunoblotting by ECL reaction using anti-ST2 (1:5000 dilution, Abcam, Cambridge, United Kingdom) and mouse anti-rabbit IgG–HRP (1:10000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GST-HRP (1:10000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-His6–HRP (1:10000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA). Gels were quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
