Cavelino 1

Whole protein lysates were prepared from transfected PC cells. Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to PVDF membranes and incubated with primary antibodies: CRT (Abcam), IRE1α (CST), PREK (CST), phosphorylation PKR-like endoplasmic reticulum kinase (p-PERK, CST), ATF-6 (CST), ZO-1 (Proteintech), ZEB1 (Proteintech), N-cadherin (Proteintech), E-cadherin (Proteintech), Vimentin (Proteintech), phosphorylation extracellular regulated protein kinases (pERK, CST), extracellular regulated protein kinases (ERK, CST), X-box-binding protein 1 (XBP1, Proteintech), Snai1 (Proteintech), Slug (CST), Cavelino-1 (Proteintech), GAPDH (Proteintech) and β-actin (Proteintech) antibodies overnight at 4 °C. Then, membranes were incubated with secondary antibodies (Santa Cruz, CA, UK) and finally detected with an ECL detection kit (Thermo Scientific, Rockford, IL, USA). The experiments were repeated for 3 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.