Protein extraction and Western blotting assay

Protein samples were prepared by homogenization of fresh human brain tissue specimen in a modified tissue lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.4], 1 mM phenyl methyl sulphonyl fluoride, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% sodium dodecyl sulphate [SDS], 5 mg/mL aprotinin and 5 mg/mL leupeptin). The tissue lysates were centrifuged for 10 min at 10,000 rpm to remove tissue debris, and the supernatants were removed and stored. The protein concentration was determined via the BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL). Glioma cells with applied treatment were also incubated with the above lysis buffer. The protein samples (30 μg per lane) were then separated by 10–12% SDS-polyacrylamide gel, and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked in 5% skim milk in TBST buffer, and was then incubated with primary and corresponding second antibodies. Immuno-reactive bands were visualized by enhanced chemiluminescence reagents (Pierce, Shanghai, China). The band intensity (in total gray) was quantified using the Image J software (NIH).