Mouse anti il 33 antibody

Total protein was extracted from 30 to 50 mg liver tissue using an extraction buffer containing RIPA lysis buffer (Thermo Fisher Scientific, 89901, Massachusetts, USA), and protease and phosphatase inhibitors cocktails (Thermo Fisher Scientific, 78441, Massachusetts, USA). A total of 50 μg protein was separated on an SDS–polyacrylamide gel with 120 V for 2 h and transferred to a PVDF membrane with 100 mA for 3 h on ice. Then, the PVDF membrane was blocked in 5% nonfat milk in TBST for 2 h at room temperature and incubated with mouse anti-IL-33 antibody using at 1:1000 dilution (Abcam, ab54385, Cambridge, England) and mouse anti-GAPDH antibody using at 1:2000 dilution (Sigma-Aldrich, G8795, St. Louis, MO, USA) overnight at 4 °C. Anti-mouse IgG (CST, 7076S, Darmstadt, Germany) was diluted at a 1:5000 ratio and used as a secondary antibody. In Fig. 7A, the secondary antibody was diluted at a 1:20,000 ratio for reacting with the primary mouse anti-IL-33 antibody. After washing 3 times with TBST, the membrane was incubated with a secondary antibody for 1 h at room temperature. After adding ECL substrate (Millipore, MA, USA), the protein band was visualized and captured with an Amersham Imager 600 system (GE, CT, USA). The exposure time was optimized as much as possible. The density of the band was quantified by Image J2x software and normalized to GAPDH.