Fndc5

BM-MSCs were lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor cocktail and insoluble debris was removed from the supernatant lysate by centrifuging at 15,000 × g for 15 min. Protein concentration was examined using BCA Protein Assay (Thermo Scientific, Waltham MA). 50 μg of total protein was loaded in each lane of 10% SDS-PAGE and transferred to nitrocellulose membranes. After the non-specific binding was blocked using 3% non-fat milk in TBST, membranes were incubated with primary antibodies against ALP (1:1000 dilutions, Abcam, UK), BMP4 (1:20000 dilutions, Abcam, UK), RUNX2 (1 µg/ml, Abcam, UK), Spp1 (1.25 µg/ml, Abcam, UK), Colla1 (1 µg/ml, Abcam, UK), FNDC5 (1:3000 dilutions, Abcam, UK), Irisin ((1:3000 dilutions, Abcam, UK), integrin αv (1:5000 dilutions, Abcam, UK) and GAPDH (1:1000 dilutions, Abcam, UK) overnight at 4 °C. Membranes were then washed three times with 0.1 M PBST, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Blots were developed and detected using Pierce ECL chemiluminescent substrates (Thermo Scientific, Waltham, MA).

To extract total proteins from cells, they were treated with a lysis buffer supplemented with protease inhibitors (Solarbio, Beijing, China, Cat. P6730), phosphatase inhibitors (Solarbio, Beijing, China, Cat. P1260) and phenylmethylsulfonyl fluoride (PMSF) (Solarbio, Beijing, China, Cat. P8340). The extracted proteins were loaded on 10% SDS-polyacrylamide gels and transferred onto nitrocellulose filter membranes (Solarbio, Beijing, China, Cat. HATF00010). Then, the membrane was rinsed with PBS and sealed with 5% skim milk in PBS for 1 h. Western blotting was performed using the standard method for the following proteins with corresponding detection antibodies (in brackets): FNDC5 (1:1000, Abcam, Cambridge, UK, Cat. ab174833), anti-ERK1/2 and anti-phospho-ERK1/2 (1:2000, Cell Signaling Technology, Boston, MA, USA, Cat. 4370S). β-actin (1:2500, Proteintech, Wuhan, China, Cat. 205361AP) was used as an internal reference. After being washed with TBST, the membranes were incubated with a secondary antibody (1:10000, LI-COR, Lincoln, NE, USA, Cat. 92632211) for one hour at room temperature. Then, the membranes were washed with TBST again and imaged with the Odyssey CLX imaging system (LI-COR, USA).