Image station 4000r pro system

To extract protein from hMSCs, the cells were lyzed using RIPA buffer composed of 50 mM Tris–HCl (pH 7.5), 0.25% Na-deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, and complete protease inhibitor cocktail (Roche, Indianapolis, IN, USA). After centrifugation at 14,000 rpm for 10 minutes, the supernatant was collected. Protein concentration was measured using the BCA Protein Assay kit (Pierce, Rockford, IL, USA). A 40-μg protein sample was loaded into each lane of a 10% polyacramide gel (Bio-Rad) for electrophoresis, and the separated proteins were then transferred from the gel onto a polyvinylidene fluoride membrane (Bio-Rad). The membrane was incubated with primary antibodies against AKT, phospho-AKT (Ser473), and glyceraldehyde 3-phosphate dehydrogenase (Cell Signaling, Danvers, MA, USA) in a blocking solution composed of Tris-buffered saline containing 5% nonfat milk (Bio-Rad) and 0.1% Tween 20 (Sigma-Aldrich) overnight at 4°C. After removing unbound antibodies, the membrane was incubated with horseradish peroxidase-linked secondary antibody (Cell Signaling) in the blocking solution for 1 hour at room temperature. The immuno-detected protein bands on the membrane were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce), and then documented by the Kodak Image Station 4000R Pro system (Kodak, Rochester, NY, USA).

Cells were lysed in RIPA buffer and then centrifuged at 14,000×g for 10 min to collect the supernatant. The protein concentration in the supernatant was measured using the BCA Protein Assay kit (Pierce, Rockford, IL, USA). A 20-μg protein sample was loaded into each lane of a 10% polyacrylamide gel (Bio–Rad, Hercules, CA, USA) for electrophoresis. Separated proteins were then transferred from the gel onto a nitrocellulose membrane (Bio–Rad). The membrane was incubated with primary antibodies against BAF60A, pSTAT3, and STAT3 (Thermo Fisher Scientific, Waltham, MA, USA) in a blocking solution composed of Tris-buffered saline containing 5% nonfat milk (Bio–Rad) and 0.1% Tween 20 (Sigma–Aldrich) overnight at 4 °C. After removing unbound antibodies, the membrane was incubated with horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, Danvers, MA, USA) in the blocking solution for 1 h at room temperature. Immuno-detected protein bands on the membrane were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and then documented by the Kodak Image Station 4000R Pro system (Kodak, Rochester, NY, USA). Information on the antibodies used in this study is provided in Supplementary Table S4.