Phospho β catenin

Cells treated with 3-DSC (0–3 μM) for 48 h in a serum free medium were homogenized with a cell lysis buffer (100 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 5 mM DTT, 0.1 mM PMSF, 10% Glycerol, protease inhibitor) and lysed with 2 h incubation on ice. The cell lysate was centrifuged at 13,000 rpm for 15 min at 4°C. Equal amounts of proteins (20 μg) were separated on a SDS/8%-polyacrylamide gel, and then transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Scientific). Blots were blocked for 1 h at room temperature with 5% (w/v) non-fat dried milk in Tris-Buffered Saline Tween-20 [TBS-T: 10 mM Tris (pH 8.0) and 150 mM NaCl solution containing 0.05% Tween-20]. After a short washing in TBST, the membranes were immunoblotted with specific antibodies. To detect target proteins, specific antibodies against stat3, stat6, phospho-stat3, phospho-stat6, β-catenin, phospho-β-catenin (1:1000, Cell Signaling Technology) and β-actin (1:5000, Sigma-Aldrich) were used. The blots were then incubated with the corresponding conjugated goat anti-rabbit or goat anti-mouse or donkey anti-goat IgG-horse-radish peroxidase (HRP) (1:5000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) secondary antibodies. Immunoreactive proteins were detected with an enhanced chemiluminescence western blotting detection system (myECL imager, Thermo Scientific).