Sc 1780

Colon tissue samples or cell samples or supernatant samples were split using RIPA assay (Beyotime) in ice. Total proteins were quantified using BCA assay (Beyotime) and were electrophoresed on 10% SDS-acrylamide gels. Total proteins were transferred to nitrocellulose membranes and membranes were blocked with 5% non-fat milk in TBS for 1 h at 37° C. Membranes were incubated with p-AMPK (ab23875, abcam), AMPK (ab32047, abcam), Nrf2 (ab62352, 1:1000, abcam), GSDMD (ab219800, 1:1000, abcam), NLRP3 (sc-66846, 1:500, Santa Cruz, USA), caspase-1 (sc-1780, 1:500, Santa Cruz, USA), and β-Actin (BS6007MH, 1:5000, Bioworld Technology, Inc.) at 4° C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5000, Santa Cruz, USA) for 1 h at 37° C after washing with TBST for 15 min. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Tissue samples or cell samples or supernatant samples were splitted using RIPA assay (Beyotime) in ice. Total proteins were quantified using BCA assay (Beyotime) and were electrophoresed on 10% SDS-acrylamide gels. Total proteins were transferred to nitrocellulose membranes, and membranes were blocked with 5% nonfat milk in TBS for 1 h at 37°C. Membranes were incubated with AdipoR1 (ab50675, 1 : 2000, abcam), AMPK (ab32047, 1 : 2000, abcam), p-AMPK (ab133448, 1 : 1000, abcam), TXNIP (ab188865, 1 : 2000, abcam), NRF2 (ab62352, 1 : 2000, abcam), HO-1 (ab52947, 1 : 2000, abcam), sOD2 (ab68155, 1 : 2000, abcam), GPX4 (ab41787, 1 : 2000, abcam), GSDMD (ab209845, 1 : 2000, abcam), NLRP3 (sc-66846, 1 : 500, Santa Cruz, USA), caspase-1 (sc-1780, 1 : 500, Santa Cruz, USA), IL-1β (sc-12742, 1 : 500, Santa Cruz, USA), and β-actin (BS6007MH, 1 : 5000, Bioworld Technology, Inc.) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1 : 5000, Santa Cruz, USA) for 1 h at 37°C after washing with TBST for 15 min. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (Bio-Rad Laboratories, Inc.).