Wnt7b

EGFR, WNT7B, β-catenin, RET, and GAPDH were purchased from Cell Signaling Technology (CST). The cells were harvested and lysed with RIPA protein extraction reagent supplemented with protease inhibitor cocktail. The protein concentrations were measured using the BCA assay (Pierce, CA, USA). Equal amounts of extracts were loaded and separated by electrophoresis on 8-10% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked for 1 h at room temperature in Tris-buffered saline/0.1% Tween 20 (TBST) containing 5% (wt/vol) nonfat milk and then incubated with primary antibodies in TBST containing 5% (wt/vol) nonfat milk or 5% (wt/vol) BSA at 4°C overnight. The membranes were then incubated with an appropriate secondary antibody coupled to horseradish peroxidase, and the proteins were detected by ECL Supersignal West Pico Chemiluminescence Kit (Thermo Fisher Scientific).