Chemiluminescence kit

NPCs were lysed with RIPA lysis buffer (Fudebio, Hangzhou, China) containing PMSF (Fudebio, Hangzhou, China) in an ice bath. After boiling with loading buffer (Fudebio), proteins from the samples were electrophoresed using SDS–PAGE gels at 80 V for 2 h and transferred to 0.22 μm polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA) at 280 mA for 120 min. The membranes were blocked with 5% skim milk powder in TBST for 2 h at room temperature and incubated with antibodies specific for β-actin (AF5001, Beyotime), MCM7 (A11325, Abclonal, Wuhan, China), p21 (27296-1-AP, Proteintech, Wuhan, China), p16 (A0262, Abclonal), aggrecan (13880-1-AP, Proteintech), collagen II (28459-1-AP, Proteintech), MMP-13 (18165-1-AP, Proteintech), and MMP-3 (17873-1-AP, Proteintech) overnight at 4 °C. The next day, the PVDF membranes were washed with TBST and incubated with an HRP-conjugated secondary antibody (Fudebio). After washing with TBST, bands were detected using a ChemiDoc Touch Imaging System (Bio–Rad, Hercules, CA, USA) with a chemiluminescence kit (Fudebio).

Total protein of mouse hindlimbs and BMSCs was extracted using the RIPA lysis buffer (CWBIOTECH, China) containing proteinase and phosphatase inhibitors (CWBIOTECH, China), and then quantified by the BCA method (FudeBio, China). The proteins were separated by SDS-PAGE gel electrophoresis (Smart-Lifesciences, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After 1 h blocking by 5% BSA, membranes were incubated at 4 °C overnight with relevant primary antibodies. The PVDF membranes were incubated in HRP conjugated secondary antibody for 1 h at room temperature. Immunoreactivities were detected by a chemiluminescence kit (FudeBio, China). Data were normalized to GAPDH and quantified by ImageJ. The antibodies used in this study were listed in Table 2.