Mab 4060

Following appropriate periods of cultivation, ESCs were washed twice with PBS and then scraped into a lysate buffer containing 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 2 mg/ml aprotinin, and 5 mM EGTA in PBS. The cells were sonicated using a sonifier cell disrupter and the resulting sonicates were centrifuged at 10,000 × g for 10 min. The supernatants were denatured in sample buffer and heated by boiling water for 5 min. The protein quantity was determined using a BCA protein assay kit (BioTeke Corporation, PP1001, China). Subsequently, the proteins were separated via 10% SDS-PAGE and electrophoretically transferred from the gels onto polyvinylidene difluoride (PVDF) transfer membranes. The membranes were blocked with 5% nonfat milk for 2 h, washed briefly in PBS-Tween, and then incubated overnight at 4 °C with primary antibodies against PTEN (1:1000, Cell Signaling, mAb #9559, USA), AKT (1:1000, Cell Signaling, mAb#4691, USA) and p-AKT (1:1000, Cell Signaling, mAb #4060, USA). An appropriate secondary antibody was applied for an additional 2-h incubation period, followed by protein expression evaluation using the Bio-Rad Imaging System (Bio-Rad Biosciences, USA), with GAPDH (1:1000, Cell Signaling, mAb #5174, USA) serving as an internal control for protein loading.