Anti mnanog

Total cell lysates were prepared using M-PER buffer (PIERCE, 78503), incubated on ice for 20 min, and cleared by centrifuging at 13,000 rpm for 10 min. Equal amounts of lysate were loaded onto 10% SDS-PAGE gels. Proteins were transferred to PVDF membranes (Bio-Rad, 1620177) using the semidry system (Bio-Rad) and then blocked with 5% milk in Tris-buffered saline–Tween 20 (TBST: 50 mM Tris, 150 mM NaCl, 0.05% Tween20) for at least 1 h at room temperature or overnight at 4°C. The following antibodies were used: anti-mNanog (R&D Systems, AF2729), anti-h/mSSEA1 (R&D Systems, MAB2156), anti-TGFBR2 (Cell Signaling, #3713), anti-IGFBP5 (R&D Systems, AF578), anti-actin (Thermo, MS1295P0), anti-AFP (Abcam, ab7751), anti-β III tubulin (R&D Systems, MAB1368), anti-WISP1 (Santa Cruz Biotechnology, sc-25441), and anti-α actinin (Sigma, A7811).