Protein Expression Analysis by Western Blot

The tissue was homogenized in radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Biotechnology, Shanghai, China) and incubated on ice for 30 min. The supernatant following centrifugation (at 15,000 × g for 15 min at 4°C) was used for western blotting. Western blotting was performed as described previously (25 (link)). Blots were incubated overnight with the following primary antibodies: Anti-podoplanin (rabbit polyclonal antibody; 1:300; cat. no. 251419; Abbiotec, San Diego, CA, USA), anti-β-tubulin (mouse monoclonal antibody; 1:5,000; cat. no. AT0003; CMCTAG, Inc., Milwaukee, WI, USA) and anti-cofilin (rabbit monoclonal antibody; 1:1,000; cat. no. 5175, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-cofilin (rabbit monoclonal antibody; 1:1,000; cat. no. 3313; Cell Signaling Technology), anti-ERM (rabbit monoclonal antibody; 1:1,000; cat. no. 3142; Cell Signaling Technology), anti-phospho-ERM (rabbit monoclonal antibody; 1:1,000; cat. no. 3726; Cell Signaling Technology), anti-MLC-2 (rabbit monoclonal antibody; 1:1,000; cat. no. 8505; Cell Signaling Technology) and anti-phospho-MLC-2 (1:1,000; cat. no. 3671; Cell Signaling Technology). Western blots were visualized using an electrophoretic gel imaging system (Shanghai, China).