Apex1

Total protein extraction was performed in tissues or cell samples, and total protein concentrations were determined. After being separated on SDS-PAGE gel, proteins were transferred to PVDF membrane (Bio-Rad) which was then blocked with 5% skimmed milk. Next, the membrane was incubated with primary antibody at 4℃ overnight and then with second antibody (Proteintech) for 1 h at room temperature. The antigen-antibody complexes were tested with ECL reagent (Monad Biotech. Co.Ltd., China). The primary antibodies were listed as follows: APEX1 (Proteintech), β-actin (Proteintech), Jagged1 (Abcam), DLL4 (Proteintech), Notch1 (Abcam), Notch3 (Abcam), RBP-JK (Proteintech), Hes1 (Abcam), Hey1 (Proteintech).

Total RNA and protein of each sample were extracted using TRIzol reagent (Ambion), and reversely transcribed into cDNA using random primers. Expression of the tested genes was quantified by qRT-PCR using 2*ChamQ Universal SYBR qPCR Master Mix and normalized to GAPDH (Roche Light Cycler). All primer sequence information is listed in Supplemental Table S1. The protein concentration was evaluated by BCA protein assay kit (Vayme Biotechnology). Primary antibodies involved in CKS2 (Abcam, cat.No: ab155078, 1:1000), APEX1 (Proteintech, cat.No: 10203-1-AP, 1:1000), APAF1 (Cell Signaling Technology, cat.No: #8969, 1:1000), PCNA (Arigo, cat.No: ARG62605, 1:5000), GAPDH ((Proteintech, cat.No: HRP-60004, 1:5000), β-Actin (Proteintech, cat.No: HRP-60008, 1:5000), E2F1 (Abcam, cat.No: ab179445, 1:1000), AKT (Cell Signaling Technology, cat.No: 4691, 1:1000), p-AKT (Cell Signaling Technology, cat.No: 4060, 1:1000), S6 (Cell Signaling Technology, cat.No: 2217, 1:1000), p-S6 (Cell Signaling Technology, cat.No: 2211, 1:1000), and PTEN (Proteintech, cat.No: 22034-1-AP, 1:1000) were used for Western blotting. Full length western blot scans for the cropped images presented in supplemental material file.