Immunoblotting analysis of epithelial-mesenchymal markers

Cells were lysed, and proteins were separated by SDS-PAGE and transferred to PVDF membrane. After blocking of non-specific binding, immunoblots were incubated with primary antibodies for Snail (L70G2; Cell Signaling Technology, Danvers, MA, USA)^{30 (link)}, E-cadherin (610182; BD Biosciences, San Jose, CA, USA)^{31 (link)}, α/β-tubulin (2148 S; Cell Signaling Technology, Danvers, MA, USA)^{32 (link)}, and GAPDH (14C10; Cell Signaling Technology, Danvers, MA, USA)^{33 (link)} followed by incubation with an anti-mouse IgG conjugated with DyLight 800 (SA5-10176; Invitrogen, Carlsbad, CA, USA)^{34 (link)} or anti-rabbit IgG antibody conjugated with DyLight 680 (35569; Invitrogen, Carlsbad, CA, USA)^{35 (link)}. Immunoblots were scanned and visualized using Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Densitometry was performed on scanned immunoblots by ImageJ.software (National Institutes of Health, Bethesda, MD, USA).