Column tissue and cell protein extraction kit

Dextran sulfate sodium (36,000–50,000 molecular weight), colitis grade, was provided by MP Biomedicals (Solon, OH, United States). Sauchinone was purchased from Med Chem Express Co., Ltd. (New Jersey, United States). Column tissue and cell protein extraction kit was provided by Epizyme Co., Ltd. (Shanghai, China). Primary antibodies in western blotting for Claudin-1, Occludin, ZO1, IL-6, TNF-α, NF-κB p65, p-IKB-α, and IKB-α were purchased from Huabio Co., Ltd. (Hangzhou, China). Primary antibodies for IL-1β, early growth response-1 (EGR1), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) were supplied by ProteinTech Co., Ltd. (Chicago, IL, United States). Anti-p-p65 and secondary antibodies were provided by Abcam Co., Ltd. (Cambridge, United Kingdom). TRIzol for RNA extraction was obtained from Thermo Fisher Co., Ltd. (Waltham, United States). The EasyScript® All-in-One First-Strand cDNA Synthesis SuperMix and TransStart® Green qPCR SuperMix were purchased from Transgene Co., Ltd. (Beijing, China). BCA protein assay kit, BeyoECL Moon, and tissue RIPA lysis buffer were provided by Beyotime Co., Ltd. (Shanghai, China).

The total protein content of heart tissues was extracted using a Column Tissue and Cell Protein Extraction Kit (EpiZyme, Shanghai, Cat #: PC201plus). The protein concentration was measured using a BCA assay and samples were incubated using Blue Loading Buffer for 30 min at 60 ℃. Equivalent amounts of samples were loaded onto an SDS-PAGE gel and then transferred to PVDF membranes (for iNOS detection, nitrocellulose membranes were used). After blocking with 5% milk in TBS with 0.1% Tween 20 for 60-90 min at room temperature, the immunoblots were incubated with the specific primary antibodies overnight at 4 ℃, and then with secondary antibodies for 60 min at room temperature. Signals were visualized using ECL-plus reagent, and images were obtained using the Bio-Rad system (Bio-Rad Laboratories, Inc.). The primary antibodies used for western blotting were: Anti‑cGAS (1:1,000, Abclonal, Cat. #: A8335), anti‑STING (1:1,000, Cell Signaling Technology, Inc., Cat. #: 13647), anti‑IRF3 (1:1,000, Cell Signaling Technology, Inc., Cat. #: 4302), anti‑phospho‑IRF3 (1:500, Cell Signaling Technology, Inc., Cat. #: 13647), anti‑iNOS (1:500, Abcam, Cat. #: ab15323) and anti‑GAPDH (1:5,000; ProteinTech Group, Inc., Cat. #: ab10494).