Gt 15094

Protein samples were isolated from differentiated cells with different drugs using RIPA buffer and transferred from the SDS-PAGE to nitrocellulose membrane (Millipore) for electroblotting. Proteins were detected with affinity-purified antibody against FoxA2 (1:1000; ab4874, Abcam), Sox17 (1:1000; GT 15094, Neuromics). Membranes were incubated in the primary antibodies overnight at 4 °C. HRP-conjugated secondary antibodies included HRP-anti rabbit IgG (1:10,000; 12-348, Millipore) and HRP-anti goat IgG (1:10,000; AP106P, Millipore). Gapdh or β-actin were used as loading controls.

mESCs were differentiated for 4 days towards definitive endoderm. The cells were washed and fixed at room temperature for 10 min in 4% Formalin solution (HT-501128, Sigma Aldrich), washed 3X with PBS and permeabilized with 0.01% triton in PBS for 1 h at RT. Immunostaining was carried out with goat antibodies against Sox17 (1:800 dilution; GT 15094; Neuromics), rabbit antibodies against FoxA2 (1:1000; ab4874, Abcam), goat antibodies against brachyury (T) (1:100; sc-17743, Santa Cruz), and mouse antibodies against Oct3/4 (1:1000; sc-5279, Santa Cruz). Fluorescence labeled secondary antibodies were Alexa Fluor 488 (green), Alexa Fluor 555 (red) and Alexa Fluor 647 (magenta) (Invitrogen, all diluted 1:1000). Nuclei were stained with DAPI (1:10,000). Images were captured using an Epifluorescence microscope (Epifluorescence, Zeiss, Germany) and analyzed with Axiovision software (Zeiss).