Western Blot Analysis of R1881 Treated Cells

Cells (400,000 cells per well) were seeded into 6-well culture plates for Western blot analysis. After 24 h, the medium was changed to a medium containing 5% FCSdcc (Thermo Fisher Scientific, Waltham, MA, USA). After another 24 h, cells were treated with different concentrations of R1881 (0.01–10 nM) for 72 h. Subsequently, cells were harvested, and protein concentration determination was performed as previously described [26 (link)]. NuPAGE^TM 4–12% Bis-Tris protein gels separated 20 µg protein lysate. As protein standard, 5 µL Spectra Multicolour Broad Range (Thermo Fisher Scientific, Waltham, MA, USA) protein standard mixed with 1 µL MagicMark^TM XP Western Protein Standard (Thermo Fisher Scientific, Waltham, MA, USA) were used. Proteins were transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (all Thermo Fischer Scientific, Waltham, MA, USA). WesternBright Sirius HRP substrate (Advansta, San Jose, CA, USA) was used to detect signals and digitalised using a Microchemi chemiluminescence system (DNR Bio-Imaging Systems, Ha-Satat, Israel). The antibodies used are listed in Table 2. Experiments were analysed with the Image-Studio Lite 5.2 software (LI-COR, Lincoln, NE, USA).