Loading buffer

The BMSCs were lysed using a cell protein extraction reagent (KEYGEN Biotech, Nanjing, China), and then mixed with loading buffer at a ratio of 4:1 (v/v) before being boiled for 5 min. The proteins were separated using 10% SDS-PAGE and subsequently transferred onto a PVDF membrane. The membrane was then blocked with blocking buffer (Thermo Fisher Scientific, MA, USA) for 10 min and incubated with the following antibodies (Table S3) overnight at 4 °C. On the second day, the bands were incubated with a secondary anti-rabbit antibody (1:3 000 BEYOTIME, Shanghai, China) for 1 h after washing with TBST. The results were visualized using an ECL chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).

Protein was extracted from harvested cells using radioimmunoprecipitation assay buffer (KeyGEN BioTECH) supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (PMSF; KeyGEN BioTECH) and phosphatase inhibitor cocktails (KeyGEN BioTECH). All the protein concentrations were detected using BCA (bicinchoninic acid) assay kit (Thermo Fisher Scientific). The same amounts of the extracted protein samples were supplemented with loading buffer (KeyGEN BioTECH) and separated in 4%–12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) gel (Sevenbio), and then transferred to a nitrocellulose membrane (Millipore, Missouri, USA). After incubation of the membranes with antibodies, the protein on the membranes was detected on an enhanced chemiluminescence (ECL) Western blotting substrate (Tanon, China). Protein membranes were analyzed by Image J software. All the experiments were conducted at least by three independent researchers.

Following the different treatments, the cells were harvested and lysed in radioimmunoprecipitation assay buffer (cat no. KGP702; KeyGen Biotech Co., Ltd.) supplemented with 1 mM phenylmethylsulfonyl fluoride (cat no. KGP610; KeyGen Biotech Co., Ltd.) and 1 mM phosphatase inhibitor cocktail (cat no. KGP602; KeyGen Biotech Co., Ltd.). The mixture was centrifuged at 12,000 × g for 20 min and the supernatant was collected. The protein concentration was determined using a BCA assay kit (cat no. KGPBCA; KeyGen Biotech Co., Ltd.) and each sample contained 30 μg protein per 10 μl. The protein samples were mixed with loading buffer (cat no. KGP101; KeyGen Biotech Co., Ltd.) and the proteins were separated using 6 or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). Following soaking in a blocking buffer (TBS with 5% nonfat dry milk) for 2 h, the blots were incubated at 4°C overnight with the primary antibody and subsequently incubated at 37°C for 1 h with the horseradish peroxidase-conjugated secondary antibody. The bands were visualized using chemiluminescence and images were captured using a ChemiDoc XRS imaging system (Bio-Rad). These were analyzed using Image Lab software (Bio-Rad).