Anti st2

IL‐33 was purchased from Novoprotein. BTZ was acquired from XIAN JANSSEN. PMA, a type of NF‐κB activators, was acquired from Sigma. NAC, a type of ROS scavengers, was procured from Selleck. Human IL‐33 neutralizing antibody (anti‐IL‐33) was purchased from R&D Systems. Anti‐β‐actin, anti‐Oct4, anti‐Sox2, anti‐c‐Myc, anti‐NF‐κB‐p65, anti‐PCNA, and corresponding secondary antibody were purchased from cell signaling technology. Anti‐ST2 was acquired from Abcam.

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).