Anti numb

Cells were harvested and lysed in RIPA buffer supplemented with protease inhibitors (50 mM Tris-HCl pH8, 50 mM NaCl, 0.5% NP-40). The protein concentrations were determined by the Bio-Rad (Bradford) protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA), and a total of 50 μg of protein was separated by denaturing 12% SDS-PAGE and transferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). After blocking with 5% nonfat milk in a 0.1% TBST solution for 1 hour at room temperature, membranes were then incubated with rabbit polyclonal anti-Numb (1:1,000; Abcam, Cambridge, UK), anti-E-cadherin (1:1,000; Cell Signaling Technology Inc., Danvers, MA, USA), anti-N-cadherin (1:1,000; Cell Signaling Technology), anti-Snail 1 (1:1,000; Abcam), anti-β-catenin (1:1,000; Abcam), and anti-β-actin (1:10,000; Abcam) overnight at 4°C. After washing, blots were incubated with HRP-conjugated goat anti-rabbit secondary antibodies (1:2,000; Abcam) for 1 hour. The immunoreactive proteins were visualized using ECL detection system (Pierce Biotechnology, Rockford, IL, USA). Protein levels were determined by normalization against β-actin. All experiments were conducted in triplicate.