Polyacrylamide gel

According to instructions of the BCA (bicinchoninic acid) protein assay (Beyotime Biotechnology Co., Shanghai, China), cell proteins were extracted to determine the protein concentration. Loading buffer was added to the extracted proteins and boiled at 95°C for 10 min. Thirty micrograms of protein sample were loaded into each well of a 10% polyacrylamide gel (Wuhan Boster Biological Technology Ltd., Wuhan, Hubei, China) and separated by electrophoresis. Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and incubated with 5% bovine serum albumin (BSA) at room temperature for 1 h. Next, the PVDF membrane was incubated with primary antibodies against, p-PI3K, PI3K, phosphorylated (p)-AKT, AKT, p-mTOR, mTOR, LC3, Beclin1, p62 and GADPH (Cell Signaling Technology, Inc.) at 1:1000 dilutions overnight at 4°C. After three washes with Tris-buffered saline containing Tween (TBST) for 5 min each, the signals on the PVDF membrane were developed by incubating the membrane with chemiluminescence reagents; GADPH was used as the internal reference. The gray value of each target band was analyzed using ImageJ software.

Proteins were extracted from tissue samples, and a bicinchoninic acid (BCA) kit (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China) was used to detect the protein concentration. After sample buffer was added (30 µg per sample each well), proteins were boiled at 95°C for 10 min. Then, the proteins were separated using 10% polyacrylamide gel (Wuhan Boster Biological Technology Co., Ltd., Wuhan, China) electrophoresis, with 80 V electrophoretic voltage converted to 120 V. After electrophoresis, proteins were transferred to polyvinylidene fluoride (PVDF) membranes with 100 V transfer-molded voltage lasting for 45 to 70 min. Afterwards, samples were incubated at room temperature for 1 h with 5% bovine serum albumin (BSA), and were incubated with primary antibodies (1: 1000 dilution) (all from Abcam Inc., Cambridge, MA, USA), overnight at 4°C. Then, samples were washed with tris-buffered saline Tween 20 (TBST) 3 times (5 min/time). The corresponding secondary antibody was added for incubation at room temperature for 1 h, after which membranes were washed 3 times (5 min/time). Development was completed with chemiluminescence reagents. GAPDH was used as an internal reference. Bands were visualized with a Bio-Rad Gel Doc EZ imager (Gel Doc EZ imager, Bio-Rad, California, USA). ImageJ software was applied to analyze the intensity of the target bands.