T mtor

EC109 and EC9706 cells were collected and total cell lysate was obtained by using RIPA buffer (ZhongShan JinQiao, Beijing, China). BCA protein assay kits (CoWin Biotechnology, Beijing, China) were used to detect the protein concentrations. Equal amounts of protein extracts from different groups were subjected to 12% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore, Boston, MA, USA). After blocking in 5% nonfat milk for 1 h, the membranes were probed with indicated primary antibodies against HMGA2, t-p70s6k, p-p70s6k, t-mTOR, p-mTOR, Ki-67, Bcl-2, Bax, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated overnight at 4°C. After washing with TBS three times, the immunoblotted membranes were further incubated with corresponding horseradish peroxidase- (HRP-) conjugated secondary antibody (Santa Cruz Biotechnology) for 2 h. Protein bands were finally visualized using chemiluminescence (Millipore, MA, USA) and normalized to β-actin.