Western Blot Protein Quantification Protocol

RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) was applied for protein extraction from cells and tissues. The lysates were then mixed with protein loading buffer (Beyotime) and denatured in the boiling water bath for 10 min. The protein products were immediately used for the expression determination as previously described [29 (link)]. Antibodies were purchased from Cell Signaling Technology (CST, Boston, MA, USA): phosphorylated (Ser 9) glycogen synthase kinase 3 beta (p-GSK3β; #5558, 1:1000), GSK3β (#12456, 1:1000), β-catenin (#8480, 1:1000), E-cadherin (#3195, 1:1000), N-cadherin (#13116, 1:1000), reference gene GAPDH (#5174, 1:1000), and Anti-rabbit IgG, HRP-linked second antibody (#7074, 1:3000). Protein blotting could be emerged using SignalFire™ Elite ECL Reagent (CST) and protein quantification was performed by ImageLab software version 4.1 (Bio-Rad).