Alpha smooth muscle actin a sma

For WB, Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA, USA) and incubated with primary CRT, pEGFR1173, pEGFR-Tyr1068 (pEGFR1068) (Abcam), pEGFR-Tyr845 (pEGFR845) (Abcam), Fibronectin, Integrinβ1, Integrinα5 (Abcam), c-Myc (Cell Signaling Technology, Danvers, MA, USA), pERK (Cell Signaling Technology), Caveolin-1 (Proteintech), E-cad, N-cadherin (N-cad, Abcam), Vimentin (Proteintech), MMP9 (Proteintech), ZO-1 (Proteintech), β-catenin (Proteintech), GATA3 (Proteintech), alpha smooth muscle actin (a-SMA, Abcam) and GAPDH (Proteintech) antibodies overnight at 4 °C. Membranes were incubated with horseradish peroxidase-conjugated monoclonal secondary antibody (Santa Cruz, CA, UK) at room temperature for 1.5 h, respectively. Immunoreactive protein bands were visualized with an ECL Detection Kit (Thermo scientific, Rockford, IL, USA). Each experiment was repeated three times.

Whole protein lysates were prepared from sg1-MSI2, sg2-MSI2, MSI2-GFP, MSI2-GFP plus MSI2siRNA and scramble transfected PC cell lines with or without EGF (50 ng/ml). Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes and incubated with primary antibodies: MSI2 (Abcam), ZEB1 (Proteintech), E-cad (Abcam), N-cadherin (N-cad, Abcam), c-Myc (Proteintech), pERK (Cell signaling technology), Cavelino-1 (Proteintech), ZO-1 (Proteintech), β-catenin (Proteintech), MMP9 (Abcam), Vimentin (Proteintech), alpha smooth muscle actin (a-SMA, Abcam), GAPDH (Proteintech), the phosphorylated epidermal growth factor receptor (EGFR) at tyrosine 845 (pEGFR845, Abcam), the phosphorylated site at tyrosine 992 (pEGFR992, Cell signaling technology) and at tyrosine 1068 (pEGFR1068, Abcam). Then, membranes were incubated with secondary antibodies (Proteintech). Protein bands were detected with an ECL detection kit (Thermol Biotech Inc., USA). Each experiment was repeated 3 times.