Bs6007mh

Cellular total protein was extracted with radioimmunoprecipitation assay buffer containing protease inhibitors. For histone extraction, cells were collected by refrigerated centrifugation at 300 × g for 10 min. The pellet was incubated for 30 min in hypotonic lysis buffer [10 mM Tris-Cl (pH 8.0), 1 mM KCl, 1.5 mM MgCl₂, 1 mM DTT and protease inhibitors]. The nuclei were resuspended in acid-extraction buffer (0.4 N H₂SO₄), and the extracted histones were precipitated with trichloroacetic acid and resuspended in deionized water. Equal amounts of protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were incubated with primary antibodies, including anti-TBX1 (ab109313, Abcam), anti-H3K4me1 (ab176877, Abcam), anti-H3K4me2 (ab32356, Abcam), anti-H3K4me3 (ab8580, Abcam), anti-histone H3 (ab176842, Abcam) and anti-β-actin (BS6007MH, Bioworld Technology), overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibodies (Sinopept) for 1 h at room temperature. Visualization was performed using an enhanced chemiluminescence detection system (Millipore). Experiments were repeated in triplicate.

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.).