Anti pgsk3b

Cells were washed with phosphate buffered solution (PBS) and whole cell lysate was prepared with modified RIPA buffer. Supernatants of the homogenates were subjected to 4%–12% Bis-Tris gel by electrophoresis, and transferred to PVDF membranes. The membranes were probed with anti-vimentin, anti-GAPDH, anti-rabbit or mouse IgG horseradish peroxidase (Sigma, St. Louis, MO, USA), anti-BMI-1 (Millipore Inc., Billerica, MA, USA), anti-AKT, anti-p-AKT (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GSK3b, anti-p-GSK3b, anti-snail, anti-α-catenin, anti-β-catenin (Cell Signaling Technology, Beverly, CA, USA) and detected with ECL Western blotting detection reagents (Thermo Fisher, Palo Alto, CA, USA).
Immunofluorescence staining was performed on cells plated in chamber slides. The cells were fixed in 3.7% formaldehyde for 15 min, washed three times with PBS and permeabilized with 0.25% Triton X-100 in PBS for 10 min. Mouse monoclonal anti-snail, anti-vimentin, and Alex Fluor®568 goat anti-mouse IgG (Millipore Inc.) were used as primary and secondary antibodies, respectively. Images were obtained using a Zeiss LSM 710 confocal microscope system (Carl Zeiss, Jena, Germany).