Enhancedchemiluminescent detection kit

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).