Proteases and phosphatases inhibitor cocktail

FF-95 cells were harvested at 1 × 10⁷ lysed with 500 µL IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) containing proteases and phosphatases inhibitor cocktail (Thermo Scientific) and then subjected to three rounds of sonication and two rounds of snap freezing in liquid nitrogen. Then, centrifugation (13,000 RPM at 4 °C) for 10 min and then collection of the supernatant were done. Protein yield was measured by Bradford assay and spectrophotometric analysis against a standard dilution. Then, 50 μg protein from each sample was run by SDS–PAGE and transferred to nitrocellulose membranes (Whatman). Membranes were blocked with 5% BSA in TBS supplemented with 0.1% Tween‐20 and incubated with primary antibodies against p21 (Cell Signaling Technology #2947), Myosin Light Chain 2 (Cell Signaling Technology #8505), Phospho-Myosin Light Chain 2 (Cell Signaling Technology #3674) overnight at 4 °C. Thereafter, membranes were incubated with secondary antibody (anti‐rabbit IgG) conjugated to HRP (Dianova, Germany) for 1 h at room temperature. Immunoreactions were detected by chemiluminescence using Vilber Fusion Fx7 system (Vilber Lourmat, Germany).