Vimentine

Cell lysis followed by protein extraction was performed as described previously (Basu et al., 2014). Lysates were subjected to immunoblotting with antibodies specific for ETS‐1, ETS‐2, MMP‐9, MMP‐13, HIF‐1α, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), glyceraldehyde‐3 phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; 1:2000); MMP‐2, protein kinase B (AKT), p‐AKT, proliferating cell nuclear antigen, E‐cadherin, N‐cadherin and vimentine (Cell Signaling, Danvers, MA, USA; 1 : 1000). Bands were visualized by reacting horseradish peroxidase‐labeled secondary antibodies (Cell signaling; 1 : 5000) with the ECL substrate (BioRad) by chemiluminesence.

BHT101 and BCPAP cells were lysed by RIPA reagent (Thermo Fisher) supplemented with protease inhibitor cocktail (Roche). Next, protein in cell lysates was separated by 4%‐10% sodium dodecyl sulfate‐polyacrylamide gel and transferred to 0.22 μm PVDF membrane (Millipore). Membrane was inhibited using 5% milk and incubated with specific antibodies at 4°C overnight. The specific band was detected by incubation with ECL chromogenic substrate and quantified by densitometry (Quantity One software; Bio‐Rad, Hercules, CA, USA). GAPDH, E‐cadherin, N‐cadherin, Vimentine, and β‐catenin antibodies were purchased from Cell Signaling Technology.