Dithiothreitol (dtt)

Fresh colon tissue was dissociated into a single-cell suspension for the detection of the T cell proportion by flow cytometry. Briefly, the tissue was cut into pieces and incubated with RPMI 1640 medium (BBI, Shanghai, China), which contained 5 mmol/L DTT (Beyotime Biotechnology, Shanghai, China), 1 mmol/L EDTA (TGREGA, Beijing, China), and 5% HyClone™ fetal bovine serum (Cytiva, United States) for 40 min at 37°C. After washing with medium twice, the colon tissue was incubated with 1 mg/mL collagenase I and 0.1 mg/mL DNase I in RPMI 1640 medium for 30 min, and the cell suspension was then filtered through a 70 μm nylon mech into a 50 ml centrifuge tube. After centrifugation at 500 g for 10 min, the supernatant was aspirated. The cell pellet was a single cell that we needed. Details of the straining method are described in an article by Han 2022.^{38 (link)} After sample preparation, a Flow Cytometer (BD Biosciences) was used for sample analysis. The FlowJo_V10 software was used for data analysis. Tregs were identified as CD45+ CD4+ FOXP3+ cells. Th17 cells were identified as CD45+ CD4+ IL17A+.
4.6 Analysis of Inflammatory cytokines
The IL10 and IL17A level in mouse serum were detected using ELISA kits (IL10:HEA056Mu, IL17A:HEA063Mu, Cloud-Clone, Wuhan, China) according to the manufacturer’s instructions.

Tyrosine phosphorylated peptides were custom synthesized by Sangon Biotech (Shanghai, China), the peptide sequence of Six2 was GEETSYCFKEKS, and the sequence of H2AX was GPKAPSGGKKATQASQRY. All peptides were purified by high-performance liquid chromatography, and all purified products were analyzed by mass spectrometry. The purity of each peptide was greater than 95%. Purified Eya1 protein (400 ng) was incubated in a final volume of 20 μL with 1 mM peptide in a buffer containing 60 mM HEPES (Beyotime Biotechnology, Shanghai, China), 75 mM NaCl (Beyotime Biotechnology, Shanghai, China), 75 mM KCl (Beyotime Biotechnology, Shanghai, China), 5 mM MgCl2 (Beyotime Biotechnology, Shanghai, China), 1 mM EDTA (Beyotime Biotechnology, Shanghai, China), and 1 mM DTT (Beyotime Biotechnology, Shanghai, China) at 37°C for 2 h. The released phosphate was quantified using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich, St. Louis, USA). Duplicate values were background corrected and compared to the internal phosphate standard.

The expression and purification of CUS_245C was described previously.¹⁶ The synthesis of the IT began with reduction of CUS_245C dimers by 0.3 mol/L DTT (Beyotime), according to the previously published method.¹⁶ Meanwhile, 25 µL of the 20 mmol/L SPDP (Thermo Fisher Scientific) solution was added to 2‐5 mg durvalumab in 1.0 mL PBS‐EDTA and the mixture was incubated for 30 minutes at room temperature. A desalting column was equilibrated with PBS‐EDTA, and the SPDP‐modified IgG was used to remove reaction byproducts and excessive nonreacted CUS_245C and SPDP reagent. CUS_245C (7.45 mg; ~5 moles CUS_245C per mole of durvalumab) was added to the durvalumab solution and the mixture was incubated at 23°C for 18 hours. The synthetic reaction was terminated by 0.1 mol/L iodoacetamide. After centrifugation and dialysis, Float‐A‐Lyzer G2 with the 100 kDa MWCO (Spectrum Laboratories) was utilized to get rid of the free CUS_245C; the synthetic product, D‐CUS_245C, was collected and purified with a Ni‐NTA column. Purified product was analyzed on 6% and 12% SDS‐PAGE gels.