Zli 2305

Total protein was extracted from tissues or cells using RIPA lysis buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein was quantified using the Pierce TM BCA Protein Assay Kit (23225, Thermo Scientific) and boiled for 10 min at 95 °C, and then equal amounts of total protein were loaded into Tris-acrylamide gels. Then the proteins were transferred to polyvinylidene fluoride (PVDF) membrane and blocked with 5% defatted milk at room temperature for 1 h. Membranes were incubated with primary antibodies at 4 °C overnight and secondary antibodies (anti-mouse IgG-HRP, ZLI-2305, Zsgb-Bio; anti-rabbit IgG-HRP antibodies, ZLI-2301, Zsgb-Bio) at room temperature for 1 h. The antibodies used in this study are listed in Table S2.

Proteins were extracted from cells using cell lysis buffer (9803, Cell Signaling Technology) containing protease inhibitor cocktail (5892970001, Merck). The following primary antibodies were used at a dilution of 1:1,000: ACTB (YM3028, Immunoway), PRPS2 (27024-1-AP, Proteintech), ADSS (16373-1-AP, Proteintech), GMPS (16376-1-AP, Proteintech), PFAS (HPA022886, Sigma‐Aldrich), GART (sc-166379, Santa Cruz Biotechnology), CAD (16617-1-AP, Proteintech), KLF4 (11880-1-AP, Proteintech), OCT4 (11263-1-AP, Proteintech), SOX2 (11064-1-AP, Proteintech), NANOG (14259-1-AP, Proteintech), VASP (13472-1-AP, Proteintech), Phospho-VASP (Ser239) (3114, Cell Signaling Technology), ERK (sc-514302, Santa Cruz Biotechnology), and P-ERK (Thr202 + Tyr204) (BS-3016R, Bioss).
The secondary antibodies anti-mouse IgG-HRP (ZLI-2305, Zsgb-Bio, dilution 1:5,000) and anti-rabbit IgG-HRP antibodies (ZLI-2301, Zsgb-Bio, dilution 1:5,000) were incubated at room temperature for 60 minutes. ECL detection reagent (WBKLS0500, Millipore) was used according to the manufacturer’s instructions