Golph3

Each cell line in logarithmic growth phase was washed with PBS. The cells were lysed in RIPA lysis buffer (containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% SDS, 1 mM EDTA, 1% Triton X-100, 1 mM PMSF, and 1 mM Protease Inhibitor Cocktail) for 20 min on ice with occasional vortex mixing. Protein concentrations were determined using the BCA assay kit (wegenebio, China). Protein concentrates of cell lysates (30 μg per lane) were separated by 10% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes (solarbio, China). Membranes were blocked with 5% skimmed milk in TBST for 1 h at room temperature. The blots were incubated with antibodies against GOLPH3 (Abcam, Cambridge, UK); pS9-GSK-3β (Cell Signaling Technology, Danvers, MA, USA); GSK-3β (Santa Cruz Biotechnology, Santa Cruz, CA, USA); β-catenin (Santa Cruz Biotechnology); β-actin (Santa Cruz Biotechnology) at 4°C overnight. After being washed three times with PBST for 10 min, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. The bands were visualized with a chemiluminescence detection kit (ECL, Rockford, IL, USA) and the result was analyzed by Image J Software. Relative intensity target protein = target band of gray value/β-actin band gradation value. β-actin was used as a loading control.