Western blotting of PI3K/AKT /mTOR signaling pathway-related proteins

After the cells in each group were harvested, lysis buffer was added to extract protein and bicinchoninic acid (BCA) method was used to measure the quantification of total protein. Then 25 μg protein was added in each well, followed by electrophoresis, transmembrane and block overnight. The membrane was washed with phosphate buffer solution tween-20 (PBST) for 5 times and added with primary antibodies (Cell Signaling Technology Inc., Beverly, MA, USA; PI3K antibody: 1: 1000; AKT antibody: 1: 1000; p-AKT antibody 1: 2000; mTOR antibody 1: 1 000; p-mTOR antibody: 1: 1 000) at 37°C for 1.5 h. After the membrane was washed for 5 times by PBS, horseradish peroxidase labeled secondary antibody was added for incubation at 37°C for 1 h. Washed in PBS for 5 times, the membrane was stained, exposed to light, developed with BIORAD GELDOC XR Gel Imaging System and fixation. Quantity One Basic software was applied for analysis of protein bands.