Horseradish peroxidase conjugated monoclonal secondary antibody

For Western Blot (WB), whole protein lysates were prepared from PC tissues and cell lines. Samples were loaded onto 10% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Millipore Corp, Bedford, MA, USA) and incubated with primary MSI2 (Abcam, Cambridge, UK) and Numb (Cell signaling technology, MA, USA) 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 hr, respectively. Immunoreactive protein bands were visualized with an ECL detection kit (ECL, Thermol Biotech Inc, USA). Each experiment was repeated three times.

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.

The total cellular and tissue protein extracts were separated on 10% SDS-polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membranes (Millipore, Bedford, MA, USA). The membranes were blocked and incubated with primary antibodies overnight at 4°C. A Bio-Rad protein assay kit was used to determine the protein concentration. The specific antibodies for p-EGFR, EGFR, p-AKT, AKT, p-ERK, ERK, and IGF-1R were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and the antibodies for p-IGF-1R and anti-GAPDH were obtained from Cell Signaling Technology (Beverly, MA, USA). GAPDH was used as a control. The membranes were incubated with the appropriate horseradish peroxidase-conjugated monoclonal secondary antibody (Santa Cruz) at room temperature for 1.5 h. An ECL detection kit (ThermolBiotech, Boston, MA, USA) was used to visualize the immunoreactive protein bands. Densitometry (Quantity One Software; Bio-Rad, Hercules, CA, USA) analysis was used to quantify the relative levels of protein expression. The experiments were repeated three times.