Anti snail1

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.

Cells were lysed using lysis buffer containing 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, 50 mM Tris (pH 8.0), and a 1:25 protease inhibitor cocktail for total protein. The protein concentration of the lysates was detected by the Bradford protein assay system (BCA; Bio-Rad). Protein samples were subject to SDS-PAGE, and transferred onto PVDF membranes. Membranes were blocked and incubated with primary antibodies at 4°C overnight. Primary antibodies, including anti-NOTCH1, anti-LDHA1, anti-E-cadherin, anti-N-cadherin, anti-Snail1, anti-TGF-β, anti-Smad3, anti-p-Smad3 were from Abcam (Cambridge, UK), and anti-actin and anti-GAPDH were from Santa Cruz Biotech (Santa Cruz, USA), and were used at a 1:1000 dilution. Then, membranes were incubated with goat anti-rabbit/mouse IgG (H+L)-HRP (Santa Cruz Biotech) secondary antibody at room temperature for 2 h. The protein signals were visualized using chemiluminescence reagent (PerkinElmer, Waltham, USA) and detected with an enhanced chemiluminescence western blotting detection system (Amersham Bioscience, London, UK).