Jagged1 fc

All information regarding antibodies used in this study is provided in Table S7. Rapamycin (Rapa), everolimus (RAD001), deferoxamine (DFX), DAPT and MHY1485 were purchased from Selleck Chemicals (Houston, TX, USA). Jagged1-Fc was obtained from R&D system (Minneapolis, MN, USA). Lipofectamine RNAiMax was obtained from Invitrogen (Carlsbad, CA, USA). pRL-TK, pGL3-Basic, pcDNA3.0, pcDNA3.0-HA-HIF-1α, lenti-CRISPRv2 plasmids and packaging vectors (pVSVG and psPAX2) were purchased from Addgene (Cambridge, MA, USA).

ESCs were plated in six-well plates at a density of 10,000 cells/cm² in K-SFM. The medium of the cells was supplemented with either Jagged1/FC (1000 ng/mL; R&D Systems, Minneapolis, MN, USA) or DAPT for interfering with the action of the Wnt/β-catenin and Notch signaling pathway. Cells in the medium with PBS were used as the control group.

Native gel electrophoresis was performed using 50 mM MES sodium buffer with 1.3% agarose (ThermoFisher Scientific, Waltham, MA, United States). Adjustments to pH were made, depending on experimental parameters. All gels were run using ice-cold buffer. Purified FGF2, Jagged1-Fc, or IgG were run individually (i.e., free) or as FGF2/IgG or FGF2/Jagged1- Fc complexes to assess their respective mobilities. Human FGF2 was cloned, expressed, and purified in our laboratory (Rao et al., 2015 (link); Miao et al., 2020 (link)). Jagged1- Fc was commercial (R&D Systems). Depending on the molar ratios for different complexes, the total protein load for each lane varied between 3.0 and 4.0 µg. After completion of runs, the gels were stained for 30 min at room temperature with Coomassie Brilliant Blue dye and de-stained overnight in 10% acetic acid in deionized water.