Cells were solubilized in lysis buffer (50 mM Tris-HCl (pH 7.4), 10% Glycerol, 1% (v/v) Triton X-100, 100 mM NaCl, 0.5 mM MgCl2, 1 mM Na3VO4, 10 μg ml−1 aprotinin, 1 mM PMSF, 1 mM pepstatin and leupeptin), and the resulting lysates were prepared by centrifugation at 16,000g for 15 min at 4 °C. The protein concentration of cell lysates was tested using the protein assay reagent (Bio-Rad) before immunoblotting. Protein samples were separated on SDS–polyacrylamide gel electrophoresis, and transferred onto PVDF membrane filters (PALL). The following primary antibodies were used according to the manufacturer's instructions: anti-phospho-Akt Ser473 (#9271, Cell Signaling), anti-Akt (#9272, Cell Signaling), anti-phospho-ERK1/2 Thr202/Tyr204 (#9101, Cell Signaling), anti-ERK1/2 (#9102, Cell Signaling), anti-NANOG (#ab21624, Abcam), anti-α-Tubulin (#T5168, Sigma) and anti-β-Actin (#sc-47778, Santa Cruz). Uncropped images of the most important immunoblots are provided in Supplementary Fig. 11.
