Dilution buffer

J1 mESCs were treated with DMSO or 3 μM CHIR for 24 h on gelatin-coated 12-well plates. The medium was discarded, and the cells were washed twice with phosphate-buffered saline before being fixed and permeabilized with immunostaining fix solution (Beyotime, Jiangsu, China) for 10 min. After blocking with immunostaining blocking buffer (Beyotime) for 1 h, the cells were incubated with primary antibody in dilution buffer (Beyotime) overnight at 4°C and then with Alexa Fluor 555-secondary antibody (Beyotime) for 2 h at room temperature in the dark. After each step, the cells were washed thrice with immunolstaining wash buffer for 5 min before the next step. DAPI (4′, 6-diamidino-2-phenylin-dole) staining was performed after secondary antibody incubation for 10 min at room temperature. The primary antibodies and dilutions used were as follows: rabbit anti-Klf4 (Abcam, Cambridge, UK; 1:500), rabbit anti-Klf4 (Boster, Wuhan, China; 1:500), mouse anti-Oct4 (Santa Cruz, CA, USA; 1:500), rabbit anti-Cdx2 (Santa Cruz; 1:500), and rabbit anti-Nanog (Cell Signaling Technology, Danvers, MA; 1:500). All reagents not indicated were purchased from the Beyotime Institute of Biotechnology (Beyotime). Immunofluorescence staining was visualized and imaged by a confocal microscope (Nikon, Tokyo, Japan).

Cell lysates were extracted from treated cells using RIPA Lysis Buffer (P0013B; Beyotime, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktail (P1045; Beyotime, Shanghai, China). Next, a bicinchoninic acid (BCA) assay (P0012; Beyotime, Shanghai, China) was performed to assess protein concentrations at 300 μg/ml and incubated with primary antibodies in dilution buffer (P0256; Beyotime, Shanghai, China) overnight at 4°C. The complexes were mixed with Protein G Agarose (10001D; Invitrogen, USA) and shaken for 3 h at 4°C to capture the antigen–antibody mixture. The beads were then washed five times with cell lysis buffer [20 mM Tris-HCl, 150 mM glycerol, 0.5% Triton X-100, 1 mM EDTA, and 1 mM ethylene glycol tetraacetic acid (EGTA)]. The eluted proteins were analyzed using the Q Exactive HF Orbitrap Mass Spectrometer (Thermo Fisher Scientific). Data were analyzed using DAVID Bioinformatics Resources version 6.8 (https://david.ncifcrf.gov/).

The alkaline phosphatase activity assay was performed according to the manufacturer’s instructions (Beyotime Biotech). Human VICs were harvested and lysed with RIPA lysis buffer without phosphatase inhibitor. Following homogenization and centrifugation, the supernatants were collected. Then supernatants were added with dilution buffer (Beyotime Biotech), p-nitrophenol (Beyotime Biotech) and the chromogenic substrate (Beyotime Biotech), next mixed using a horizontal shaker, and incubated at 37°C for 20 min. The absorbance at 405 nm was detected using an EPOCH2 microplate reader (Thermo Fisher Scientific) to measure alkaline phosphatase activity.