2.14. Western blot analysis

Total protein of the U87 and U251 cells was extracted using radio‐immunoprecipitation assay (RIPA) lysis buffer (R0010, Solarbio Life Science), with protein concentration measured using a bicinchoninic acid (BCA) protein assay kit (G3522‐1, GBCBIO Technologies). After polyacrylamide gel electrophoresis (PAGE), the protein was transferred onto nitrocellulose membranes using the wet transfer method and then blocked with 5% BSA for 1 hour at room temperature. The membranes were incubated with the following primary antibodies to LC3 (1:500, ab128025), Beclin1 (1:1000, ab62557), Bax (1:1000, ab53154), Bcl‐2 (1:1000, ab59348), Caspase‐3 (1:500, ab13847) and PKC (1:1000, ab19031) at 4°C overnight. All antibodies were purchased from Abcam. Next, the membranes were washed five times with PBS (5 minutes each time) and incubated with horseradish peroxidase (HRP)‐labelled secondary rabbit anti‐human antibody to IgG (1:5000, F020218, Baiaolaibo Science and Technology Ltd.) for 1 hour. Each membrane was developed using electrochemiluminescence (ECL) (ECL808‐25, Biomiga) for 1 minutes. After the solution was discarded, each membrane was covered by preservative film and exposed by X‐ray (36209ES01, Shanghai qcbio Science & Technologies, Co., Ltd.) in order to visualize the results. GAPDH (1:1000, ab8245, Abcam) was used as the internal reference and relative expression was computed as the ratio of the grey value of the target band to that of the internal reference band.