RASSF1 Western Blot Analysis

Whole‐cell lysates were obtained from RASSF1 and control shRNA transfected HEK293FT cells treated with tunicamycin (5 μg·mL⁻¹) for 5 h. The cells were harvested with RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA). Lysates were sonicated for 30 s, and the protein concentration was measured by DC protein assay (Bio‐Rad). Protein samples (30 μg each) were separated by 4–12% PAGE Gel (GenScript, Piscataway, NJ, USA) and then transferred onto PVDF membranes (Millipore‐Sigma, Burlington, MA, USA). Membranes were blocked with 5% nonfat milk for 60 min at room temperature and incubated overnight at 4 °C with recombinant anti‐RASSF1 rabbit monoclonal antibody (1 : 500, ab126764; Abcam) or anti‐α‐Tubulin monoclonal mouse antibody (1 : 5000, T9026; Sigma). After washing, membranes were incubated for 1 h at room temperature with horse radish peroxidase (HRP) conjugated goat anti‐rabbit IgG secondary antibody (1 : 10 000; Abcam) or goat anti‐mouse IgG secondary antibody (1 : 10 000; ThermoFisher) at room temperature. The immobilized proteins were detected using the enhanced chemiluminescence reagent plus (PerkinElmer, Waltham, MA, USA). Images were obtained with ChemiDoc™ Touch Imaging System (Bio‐Rad) and analyzed with image lab.