Af410

Proteins were extracted from colon tissues using RIPA lysis buffer supplemented with proteinase and phosphatase inhibitors (Roche) as previously described.³⁰ . Protein concentration was assessed using Pierce BCA protein assay (ThermoFischer Scientific) and all samples were normalized to 2μg/ml. Samples were resolved by 8%–15% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE) electrophoresis and transferred onto polyvinylidene difluoride membranes. Blocking was performed in 5% skim milk for 1 h, and membranes were incubated with primary antibodies overnight at 4°C. Membranes were incubated with horse radish peroxidase–conjugated secondary antibody for 1 h, and proteins were visualized by using ECL substrate (ThermoScientific). Primary antibodies used were anti-pyrin (1:1000, ab195975, Abcam), anti-caspase-1 (1:500, sc-515, Santa Cruz Biotechnology), anti-P-STAT3 Tyr705 (1:1,000, 9131, Cell Signaling Technology), anti-IL-1β (1:1000, AF410, R&D systems) and anti-GAPDH (1:10,000; 5174, Cell Signaling Technology).