Phosphorylated MLC of TM and SCE was determined by Western blotting. The cells were cultured on 6-cm dishes, and treated with 100 nM DEX and 10 μM Y-27632 (Merck KGaA, Darmstadt, Germany) for 5, 15, or 30 minutes. As the positive control, monkey TM cells were treated with 20 μM lysophosphatidic acid (LPA; Sigma-Aldrich, St. Louis, MO) for 30 minutes. Cells then were washed with ice-cold PBS and lysed in 300 μL RIPA buffer (Thermo Fisher Scientific, Waltham, MA) containing a protease inhibitor (Thermo Fisher Scientific) and a phosphatase inhibitor (Nacalai Tesque, Kyoto, Japan). The cell lysates were sonicated and centrifuged at 15,000 revolutions per minute (rpm) for 10 minutes at 4°C. Protein concentrations of the cell lysates were measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). The cell lysate was mixed with NuPAGE LDS sample buffer and 50 mM dithiothreitol (Life Technologies, Carlsbad, CA) and heated at 70°C for 10 minutes. Equal amounts of the protein samples were loaded onto a 10% or 4% to 12% gradient polyacrylamide gel (Life Technologies), and proteins were fractioned by SDS-PAGE. The separated proteins were transferred to PVDF membranes. Membranes were blocked with 2% blocking reagent (GE Healthcare) in Tris-buffered saline (137 mM NaCl, 20 mM Tris, pH 7.4) containing 0.1% Tween-20 (TBS-T) for 60 minutes at room temperature, and then incubated overnight at 4°C with rabbit polyclonal antibody phospho-MLC or total MLC (1:1000 dilution in 5% bovine serum albumin/TBS-T; Cell Signaling Technology, Danvers, MA). Membranes were washed 3 times for 10 minutes each with TBS-T, incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000 dilution in TBS-T; Cell Signaling Technology) for 30 minutes at room temperature and visualized using ECL Advance Western Blotting Detection Reagent (GE Healthcare). All membranes were stripped of antibodies using WB Stripping solution (Nacalai Tesque) and incubated with mouse monoclonal antibody β-actin (1:10,000 dilution in blocking solution; Sigma-Aldrich), and subsequently with HRP-conjugated goat anti-mouse IgG (1:20,000 dilution in TBS-T; GE Healthcare) for a loading control. β-Actin immunoreactive bands were visualized with ECL Western Blotting Detection Reagent (GE Healthcare) and determined using a luminescent image analyzer.