ChIP assays were performed using ChIP-IT High Sensitivity Kits (Active Motif). Specifically, 5 × 105 CD8+ Tregs were cross-linked with Complete Fix buffer for 30 minutes at room temperature and quenched with Quench buffer for 5 minutes. Cells were resuspended with ChIP Prep buffer and nuclei were extracted with a Dounce homogenizer, resuspended with ChIP buffer, and sonicated into yield chromatin fragments of 300 to 1000 bp. Lysates were incubated with Protein G beads and immunoprecipitated with 8 μg of anti-NOTCH4 (Cell Signaling Technology, 2423) or anti-HES5 antibody (Abcam, ab194111) overnight at 4°C. Proteins were washed and eluted from Protein G beads, followed by incubation with RNase A and proteinase K. DNA was cleaned using a PCR purification kit (Qiagen) and promoter regions of RAB5A, RAB7A, and RAB11A were amplified by qPCR via specific primers. All primer pairs are included in Table 3.

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