P akt 4060 t

Western blotting was carried out as described previously [24 (link)]. In brief, for Western blotting, proteins were extracted using RIPA buffer mixed with protease inhibitors (1:100), phenylmethylsulfonylfluoride (PMSF, 1:100) and dithiothreitol (DTT, 1:100) at 4 °C for 30 min. The lysate was centrifuged at 13000 rpm at 4 °C for 15 min. The supernatants were collected and protein concentrations were measured using the BCA method. For Western blot analysis, protein samples were separated by 8% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad). The membranes were then blocked with 5% nonfat milk and incubated with primary antibodies overnight followed by washing and reaction with horseradish peroxidase–conjugated secondary antibody for 1 h at 37 °C. The reaction was detected by using ECL reagents (GE Healthcare) and the signals were captured by X-ray films. Antibodies against RIF1 (sc-515,573) and β-actin (sc-58,673) were obtained from Santa Cruz Biotechnology. Antibodies against NANOG (ab21624) and SOX2 (ab97959) were purchased from Abcam. Antibodies against PI3K (4257 T), p-PI3K (4228 T), AKT (4691 T) and p-AKT (4060 T) were purchased from Cell Signaling Technology. Antibody to hTERT (AP1410d) was purchased from Abgent.

Primary antibodies against AKT (#4685S), p-AKT (#4060T), β-catenin (#8480T), CD63 (#55051S), CD81 (#56039) and CD9 (#13403) were purchased from Cell Signaling Technology, USA. The HRP-labeled anti-rabbit IgG monoclonal antibody (#D110065, Sangon Biotech, China) was used as the secondary antibody, and the fluorescent signal was developed with ECL substrate (#SW2010, Solarbio, China). The chemiluminescent signals were captured using a Bio-Rad ChemiDoc XRS system (Bio-Rad, USA).