Enhanced chemiluminescence detection kit

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).

The cells were lysed in RIPA buffer (Beyotime, Jiangsu, China) with PMSF and phosphatase inhibitor cocktail (Fudebio, Hangzhou, China), and the lysates were subjected to centrifugation at 12,000 × g for 10 min. The protein concentration of the supernatants was measured with the BCA kit (Beyotime, Jiangsu, China). The samples were resolved using SDS-PAGE and then transferred onto PVDF membranes (EMD Millipore, Billerica, Massachusetts, USA). The unwanted interactions were blocked with 5% skim milk for 120 min. The blocked membranes were incubated with corresponding primary antibodies targeting TRADD, FADD, RIPK1, cCASP3, ZO-1, or GAPDH (1:1,000; Affinity, USA) overnight at 4°C, followed by secondary antibodies (1:5,000; Proteintech, China) at room temperature (RT) for 60 min. Lastly, detection was performed using the enhanced chemiluminescence detection kit (Yeasen, Shanghai, China).

Cells were collected at the specified times and washed twice with PBS. The cell extracts were prepared using lysis buffer and then centrifuged at 13,000 × g for 30 minutes at 4 °C. The protein concentration was quantified using a BCA kit (Yeasen, Shanghai, China). The supernatant was collected, and 5× SDS protein loading buffer was added in proportion, followed by incubation at 100 °C for 10 minutes. Proteins were separated on a 10–12% SDS‒PAGE gel and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Boston, MA). The membranes were blocked with 5% nonfat milk in Tris‐buffered saline supplemented with 0.1% Tween 20 at room temperature for 2 hours. The primary antibody was diluted with 1× TBST according to the instructions and incubated overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies for another 2 hours at room temperature. Immunoblots were visualized using an enhanced chemiluminescence detection kit (Yeasen, Shanghai, China) with a chemiluminescence imaging analysis system (Tanon, Shanghai, China). Relative integrated density values were calculated using ImageJ software. The following antibodies were used: anti-cGAS, cGAS (E5V3W); rabbit mAb #79978 (CST, Boston, MA); anti-NF-κB, p65 (D14E12); XP® rabbit mAb #8242 (CST, Boston, MA); and GAPDH (Sigma‒Aldrich, Louis, MI).