A p s6k1

The IF of paraffin sections was carried out as described previously (Qin et al., 2019) . After deparaffinization, the sections were blocked with 5% goat serum at RT for 45 minutes, then incubating with primary antibody (a-Oct4, Abcam, ab181557; a-p-S6K1, Sigma, SAB4301518) at RT for 1 h. After washing in 1×PBS buffer for three times, the slides were incubated with Alexa Fluor 594 conjugated goat anti-rabbit IgG (Invitrogen, A11037) at RT for 1 h. The nucleus was stained with DAPI (Thermo Fisher). All images were captured with Leica DM3000 microscope with DFC420C camera.

ESCs were collected and dissociated in cold lysis buffer (20 mM HEPES, pH 7.7; 5 mM KCl; 1.5 mM MgCl2; 2 mM DTT; 2 mM PMSF) for 30 minutes on ice. Then the cell lysate was centrifuged at 12,000 rpm, 15 minutes at 4 ℃. Protein concentration was measured using BCA Protein Assay Kit (Beyotime). The samples were resolved with SDS-PAGE gel and transferred onto PVDF membrane. Then, the PVDF membrane was incubated in blocking buffer for 1 hour at RT, followed by incubating with primary antibodies (a-Hbxip, CST, 14633S; a-Nanog, Bethyl Laboratories, A300-397A; a-Oct4, Santa Cruz, sc-5279; a-Tubulin, Sigma, ASB4800087; a-S6K1, Sigma, SAB4502686; a-p-S6K1, Sigma, SAB4301518; a-H3, Abcam, ab1791) at 4 ℃ overnight. After washing three times, the membrane was incubated with HRPliked secondary antibodies at RT for 1 hour. Immunoreactivity was detected by ECL Plus (Beyotime). Digital images were taken with by the automatic chemiluminescence imaging analysis system (Tanon).