Prdm1

The expression of BMP4 and its receptors as well as germ cell-specific markers in EB cells was obtained by immunocytochemistry according to the procedure described previously (Liu et al. 2015) (link). Briefly, iPS cells were fixed in 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton X-100. The cells were blocked with 10% serum and incubated with primary antibodies against BMP4 (Abcam), BMPR1A (Santa Cruz), BMPR1B (Santa Cruz), BMPR2 (Santa Cruz), PRDM1 (Abnova, Taipei, Taiwan) and VASA (Abcam) overnight at 4°C. The detailed information on antibodies was shown in Table 3. The cells were then incubated by goat anti-rabbit Alexa Fluor 594 (red)-labeled secondary antibody (Invitrogen) for 1 h. DAPI was used to label cell nuclei, and images were captured with a fluorescence microscope (Leica).

The EBs derived from iPS cells were lysed using the RIPA buffer (Santa Cruz) containing a cocktail of protease inhibitors (Roche). Cell lysates were cleared by centrifugation at 12,000 g, and the concentrations of total proteins were measured by BCA kit (Dingguo Company, Beijing, China). Twenty micrograms of total protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% BSA and 0.1% Tween20 for 1 h at room temperature, membranes were probed with primary antibodies, including PRDM1 (Abnova), VASA (Abcam), DAZL (Abcam), UCHL1 (AbD Serotec Kidlington, UK), GFRA1 (Santa Cruz), KIT (Abcam), phospho-Smad1/5 (Cell signaling), Smad5 (Cell signaling) and ACTB (beta-actin, Proteintech) overnight at 4°C. The detailed information on antibodies was shown in Table 3. The blots were incubated with HRP-conjugated antirabbit or anti-goat IgG polyclonal secondary antibodies (Santa Cruz) at 1:2500 dilution for 1 h at room temperature. After extensive washes with TBST, the blots were visualized using an enhanced-chemiluminescent detection kit (Santa Cruz).