Western Blot Protein Analysis in U87 Cells

The assay was performed as previously described (18 (link)). Briefly, total protein was extracted from the U87 cells and concentration was measured using the bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. Protein samples (100 µg) were run on an SDS-polyacrylamide gel (3 h at 70 V) and the proteins were then transferred to nitrocellulose membranes (Sigma-Aldrich). The membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. This was followed by incubation with relevant secondary antibodies (1:10,000; Abcam, Cambridge, UK) for 1 h at room temperature. The bands were visualized using a ChemiDoc MP Imaging System (Bio-Rad Laboratories, Inc. (model 1708280; Hercules, CA, USA).