Ecl stripping buffer

Cells were lysed in the presence of 100 µl radio-immunoprecipitation (RIPA) buffer containing protease inhibitors (Alfa Aesar, Stoughton, MA, USA) on ice. Protein was quantified using the Bradford Assay (BioRad, Hercules, CA, USA). Twenty micrograms of total protein from each sample was resolved on a 4–12% SDS-PAGE gel and transferred to PVDF membranes. Sister blots were then probed with antibodies including anti-E-cadherin (Cell Signaling, Danvers, MA, USA), anti-vimentin (Protein Technologies, Tucson, AZ, USA), or anti-β-actin (BD Biosciences, San Jose, CA) antibody. Chemiluminescent signals were detected with ECL™ prime (Thermo Fisher Scientific) using the Biorad ChemiDoc system. If necessary, blots were stripped with ECL Stripping Buffer (Li-Cor, Lincoln, NB, USA) following manufacturer protocol. Bands were quantified using ImageJ.

Cells were lysed in ice-cold radio-immunoprecipitation (RIPA) buffer containing protease inhibitor and phosphatase inhibitor cocktail (Alfa Aesar, Stoughton, MA, USA). Protein was quantified using the Bradford Assay (BioRad, Hercules, CA, USA). Proteins (20–30 μg) were resolved in SDS-polyacrylamide gels (10%) and transferred to PVDF membranes using a Tris-glycine buffer system. Membranes were blocked with 5% non-fat dry milk in 0.1% Tween20 in TBS (TBST) for 1 h at room temperature followed by probing with primary antibodies and gently rocking overnight at 4 °C. Antibodies used for immunoblotting were Mouse anti-E-cadherin (Cell Signaling, Danvers, MA, USA), Rabbit anti-N-cadherin (Cell Signaling), Rabbit anti-vimentin (Protein Technologies, Tucson, AZ, USA), Rabbit anti-survivin (Cell Signaling), Rabbit anti-Lasp1 (Cell Signaling), Rabbit anti-BMI1 (Cell Signaling), and Mouse anti-β-actin (BD Biosciences, San Jose, CA, USA). The next day, the blots were washed with TBST three times, 5 min each time. They were incubated with secondary antibodies (1:2000) at room temperature for 1 h. Chemiluminescent signals were detected with ECL™ prime (Thermo Fisher Scientific, Waltham, MA, USA) using the Biorad ChemiDoc system. If necessary, blots were stripped with ECL Stripping Buffer (Li-Cor, Lincoln, NB, USA) following the manufacturer’s protocol. Bands were quantified using ImageJ.