Protein Expression Analysis in Cells

Cytoplasmic and nuclear proteins were extracted with an NEPER Nuclear and Cytoplasmic Extract Kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions. Aliquots of cell lysates were separated using a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and then electroblotted onto nitrocellulose membranes (Bio-Rad), as previously described (Xie et al., 2014 (link)). The membranes were incubated with antibodies raised against ENO1 (#ab155102) and β-catenin (#ab32572) (both from Abcam, San Francisco, CA, USA), and those recognizing β-actin (#4970), N-cadherin (#13116), E-cadherin (#3195), snail family transcriptional repressor 2 (SLUG) (#9585), Vimentin (#5741), MMP-2 (#40994), MMP-9 (#13667), lamin A (#86846), and snail family transcriptional repressor (SNAIL) (#3879) (all from Cell Signaling Technology, Beverly, MA, USA) overnight, followed by the addition of horseradish peroxidase-linked anti-rabbit/mouse immunoglobulin G (IgG) (Abcam #ab205718/ab205719) and enhanced chemiluminescence visualization of the immunoreactive protein bands.