Protein Expression Analysis in Cell Lysates

Following treatments, total cell lysates were prepared using previously described methods [47 (link)]. Primary antibodies against pIGF1R (Tyr1135/1136), IGF1R-β, Snail-1, claudin-1, and ZO-1 were from Cell Signaling Technology, Inc. (Beverly, MA, USA). ZEB-1 antibody was from Santa Cruz Biotechnology, Inc. Antibodies against β-actin, and vimentin purchased from Sigma (Saint Louis, MO, USA) and pFAK (Tyr397), total FAK, and E-cadherin from BD Biosciences were used for Western blots according to standard protocol. Bound primary antibodies were detected with peroxidase-coupled secondary antibodies (Southern BioTech; Birmingham, AL, USA) and developed by enhanced chemiluminescence (Luminata Classico Western HRP substrate; EMD Millipore Corp.; Billerica, MA, USA).