Protease and phosphatase inhibitors

Protein extracts were obtained from cells and rat duodenal tissue samples employing total lysis buffer (Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitors (Fudebio, Hangzhou, China). After homogenization and centrifugation at 14,000 rpm for 15 min at 4 °C, concentration of protein supernatant was measured using a standard Bradford assay (ThermoFisher Scientific, Rockford, USA). The samples were resolved using SDS-PAGE, then transferred onto PVDF membranes (EMD Millipore, Billerica, Massachusetts, USA), and blocked with 5% skim milk for 120 min. The blocked membranes were incubated at 4 °C overnight with corresponding primary antibodies targeting GSDMD full length and cleaved GSDMD (C Teminal) (Abcam, USA), Caspase-1 (Proteintech, Wuhan, China), IL-1β (Abcam, USA), β-Tublin (Abmart, Shanghai, China), Cleaved Caspase-1 (CST, USA)) followed by secondary antibodies (Proteintech, Wuhan, China) at room temperature for 120 min. Lastly, the detection was performed using the enhanced chemiluminescence detection kit (Yeasen, Shanghai, China).

Cell lysates or intestine homogenates was prepared with RIPA buffer, protease and phosphatase inhibitors (Fudebio, Hangzhou, China). After SDS-PAGE separation, PVDF membranes were incubated with specific antibodies as described above followed by probed with HRP-conjugated goat anti-mouse or anti-rabbit IgG.

Total cellular proteins were extracted with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Fudebio, Hangzhou, China). The concentration of total proteins was determined with a BCA kit (Thermo, USA). Total proteins (20 µg) were electrophoresed on 10% SDS-PAGE, then electro-transferred to PVDF membrane (Millipore, USA). The membrane was cut into strips and incubated individually with the primary antibodies at 4°C overnight, followed by incubation with the secondary antibodies for 1 h at room temperature. The bands were visualized with enhanced chemiluminescence system (Millipore, USA). The corresponding antibodies are listed in Table S3.