Western Blot Assay Protocol

The Western blot assay was executed as previously described [49 (link)]: 1 × 10⁶ cells were seeded in Petri dishes (ø 6 cm, 5 mL/dish) and incubated overnight. On the next day, the cells were treated with the compounds in fresh culture media (5 mL/dish) for the indicated time. Next, cells were harvested with a cell scraper and lysed in lysis buffer including protease and phosphatase inhibitors cocktail (Roche, Mannheim, Germany). Cell debris were removed from the protein lysates via centrifugation. Afterwards, the cell-free protein mixtures were separated in gradient ready-made Mini-PROTEAN^® TGX Stain-Free^TM gels (Bio-Rad, Hercules, CA, USA) using SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis). Then, the proteins were blotted onto ø 0.2 µm pore PVDF membrane. The membrane was blocked and consequently treated with primary and secondary antibodies for protein detection. The signals were detected using the ECL chemiluminescence system (Thermo Scientific, Rockford, IL, USA). The antibodies used are listed in Table 2.