Western Blotting of Cell Signaling Proteins

Western blotting was performed as previously described [6 (link)]. Briefly, total protein concentration in the cell lysate was determined by using DC™ protein assay (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of total protein were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to a nitrocellulose membrane. The membrane was incubated with blocking buffer (Thermo Fisher) for 1 h at room temperature and incubated overnight at 4°C with the primary antibody to SNAIL1, E-cadherin (1:1000 dilution, Cell Signaling), IGF1R (1:1000 dilution, Abcam, Cambridge, MA, USA), CD9, CD63 or HSP70 (1:1000 dilution, SBI System Biosciences, Mountain View, CA). The membrane was washed with Tris-buffered saline-Tween-20 buffer (TBST) and incubated with HRP-conjugated secondary antibodies (1:10,000 dilution, Abcam) at room temperature for 1h. After washing with TBST immunoreactivity was detected using enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences, Marlborough, MA). β -actin, the loading control, was detected using β -actin antibody (1:10000, Abcam) and anti-mouse IGG (1:10000, Sigma Aldrich) as the primary and secondary antibodies, respectively.