Pierce s halt protease inhibitor cocktail

Cell lysates were obtained by extraction in RIPA buffer (20 mmol/L Tris-HCl, pH7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 1% NP-40) containing Pierce’s Halt Protease Inhibitor Cocktail (Thermo Scientific). The protein concentration was determined by Bradford’s assay using a Bio-Rad protein assay kit (Bio-Rad). The proteins (40 μg) were separated on a 12% SDS-polyacrylamide gel and then transferred to a PVDF membrane. Western blot analysis was performed using antibodies (1:1000 dilution, Cell Signaling Technology, Inc.) against phospho-Erk 1/2 (#4370), phosphor-Akt (#4060), p-Smad2 (#3101), E-cadherin (#3195), Snail (#3879), vimentin (#5741), and vitamin D receptor (#12550), or antibodies against Zeb1 (1:1000 dilution, sc-25388,Santa Cruz Biotechnology), orCYP24A1 (1:1000 dilution, ab 109632, Abcam).

Cell lysates were obtained by extraction in RIPA buffer (20 mm Tris-HCl, pH7.4, 150 mm NaCl, 5 mm EDTA, 1% NP-40) containing Pierce’s Halt Protease Inhibitor Cocktail (Thermo Scientific, Rockford, IL, USA). Protein concentration was determined by Bradford’s assay using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Proteins (40 μg) were loaded onto a 12% sodium dodecyl sulfate–polyacrylamide gel. Proteins were transferred to a polyvinyl difluoride membrane and subjected to western blot analysis using the following antibodies from either Cell Signaling Technology (Boston, MA, USA): p53 (cat# 2524), phospho-ERK1/2 (cat# 4370), E-Cadherin (cat# 3195), phospho-AKT (cat# 4060), total-ERK1/2 (cat# 9102), total-AKT (cat# 9272) and β-actin (cat# 4970) or Santa Cruz Biotechnology (Dallas, TX, USA): p16^INK4A (cat# sc-1207) and p21^CIP1/WAF1 (cat# sc-397). All antibodies were used at 1:1000 dilutions except for total-ERK1/2, total-AKT and β-actin where 1:5000 dilutions were used. Quantification of western blots was performed using ImageJ software (http://rsb.info.nih.gov/ij/).