Acrylamide bis solution

Cells were lysed in sample buffer (2% SDS, 0.1 mg/ml bromophenol blue, Tris-HCl pH 6.8, 5% 2-mercaptoethanol and 10% glycerol), and boiled at 100°C for 5 min. For the one of experimental group, each cells of 6-well plate were recovered with 200 μl sample buffer and accumulate. Protein was quantified using the bicinchoninic acid (BCA) (Thermo Scientific) assay after addition of sample buffer. The samples were loaded on 10% SDS-polyacrylamide gel electrophoresis (3.3 ml of 30% acrylamide/Bis Solution (BIO-RAD, CA, USA), 2.5 ml of 1.5 M Tris Glycine buffer (pH 8.8, Dyne Bio, Korea, Gyeonggi-do), 0.1 ml of 10% SDS solution (Dyne Bio, Korea, Gyeonggi-do), 0.1 ml of 10% ammonium persulfate solution (Dyne Bio, Korea, Gyeonggi-do), 4 ul of N,N,N’,N’-Tetramethylethylenediamine (Biosesang, Korea, Gyeonggi-do) and 4.0 ml of H₂O), then the separated proteins were transferred to a nitrocellulose membrane (BIO-RAD, CA, USA), and then incubated with primary antibodies recognizing β-actin (Sigma–Aldrich, St Louis, USA), type I collagen, fibronectin, β-catenin, wnt3a and interleukin-1α (IL-1α) (ABCAm, Massachusetts, USA). Protein detection was performed with Lumi Femto (Daeil Laboratory Service, Seoul, Korea) and the Molecular Imager ChemiDoc XRS+ station. Image J software (National Institutes of Health, Bethesda, MD, USA) was used to analyze and quantify the western blot image.